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CONSTRUCTION OF AN INDUCmLE VECTOR CONTAINING MARKER GENE
Nurul Ashikin Mohamed Ali
OK 9814
Bachelor of Science with HonoursN974 (Resource Biotechnology)
2005
2006
lu~al Khidrn~1 MaklllInat A_kndemB tJNlVERSrrr MALAYStA SARAWAK
9j 1()(1 yen Ow ~mahan
P_KHIDMA TMA KLUMA TAKADEMIK UNIMAS
111111111 111 1111111111111 1000127070
CONSTRUCTION OF AN INDUCIBLE VECTOR CONTAINING MARKER GENE
NURUL ASHIKrN MOHAMED ALl
This project is submitted in partial fulfillment of the requirements for the degree of Bachelor o f Science with Honours
(Resource Biotechnology)
Faculty o f Resource Science and Technology UNIVERSITY MALAYSIA SARAWA K
2005
CONTENTS
Content Page
Acknowledgement lV
Li st of Figure v
List ofTable Vl
Abstract Vll
Chapter 1 INTRODUCTION
11 Backgrollnd
111 Transfolmation
112 Restri ction enzyme
113 Polymerase Chain Reaction 2
114 Ligation 3
115 Plasmid 4
116 Reporter gene 9
117 Inducibl e promoter 10
118 Heat shock inducible promoter II
119 Tetracycline inducible promoter II
1110 Ethanol inducible promoter 12
12 Research Objectives 14
CONTENTS
Content Page
Chapter 2 MATERIAL AND METHOD
21 Preparation ofOvemight BacteIial Culture for Preparation of 15
Competent Cell s
22 CaCh bacteria l competent cells preparati on 15
23 Bacterial plasmid transformation 16
24 Mini-Prep Iso lation of Double Stranded Plasmid DNA from 16
Bacterial Culture
25 Restriction Enzyme Analysis 17
26 Agarose gel electrophoresis 18
27 Polymerase Chain reac tion 19
28 DNA extraction from agarose gel 2 1
29 Calf Intestine Alkaline Phosphate 21
210 Ligation 22
11
CO TENTS
Content Page
Chapter 3 RESULT AND DISCUSSION
3 1 Bacterial plasmid transfOlmation 23
32 Mini prep plasmid isolation 23
33 Restriction enzyme 24
34 Polymerase Chain Reaction 29
35 Ligation 3 1
Chapter 4 CONCLUS ION AND RECOMMENDATION 34
REFERENCES 35
APPENDIX 38
III
Acknowledgement
I would like to express my sincere app rec iation to my supervisor Dr Hairul
Azman Roslan for hi s guidance throughout this project A special thanks to my family
master students and my fe llow friends for their explanation and help during this project
IV
LIST OF FIGURE
Content Page
Figure I Restri ction maps ofpAGS 4
Figure 2 Restriction maps of pALS 5
Figure 3 Restriction maps ofpSRN 6
Figure 4 Restriction maps ofpGPTV 7
Figure 5 Graph expression of inducible gene JI
Figure 6 Mechanism of induc ible promoter 12
Figure 7 Plasmid iso lation 23
Figure 8 Restriction enzyme digestion 25
Figure 9 Restriction enzyme digest ofpAGS and pALS with HindIlI 26
Figure 10 Restriction enzyme digest pSRN and pGPTV with Hindlll 27
Figure 11 Result ofPCR to detennine the genotype of p lasmid 28
Figure 12 Plasmid isolation after digestion 29
Figure 13 Result of PCR 30
Figure 14 Possible orientation 31
v
LIST OF TABLE
Content Page
Table I List the reagent and volume of restriction enzyme ana lysis 16
Table 2 Lists the amount and volume used in PCR reaction 18
Table 3 List the reagent and vo lume used to prepared PCR reaction 19
using premix Qiagen PCR mixture
Table 4 PCR cycle used in routine determination of GUS LUC and 19
al cR gene in the plasmids
Table 5 The sequences of primers used for PCR work 20
Table 6 List the reagent and volume used to prepared PCR reaction usin g 2 1
premix Qiagen PCR mixtu re
Table 7 Lists the reagent and vo lume llsed for ligation process 21
Table 8 Shows the pl as mids restriction enzyme used to detect the insert 24
and the size of insert afteT digeslon
Table 9 Combination of pIimers used to detect the plasmid using PCR 28
Table 10 Combination ofprimern used to detec t the plasmid ll sing PCR 30
VI
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
lu~al Khidrn~1 MaklllInat A_kndemB tJNlVERSrrr MALAYStA SARAWAK
9j 1()(1 yen Ow ~mahan
P_KHIDMA TMA KLUMA TAKADEMIK UNIMAS
111111111 111 1111111111111 1000127070
CONSTRUCTION OF AN INDUCIBLE VECTOR CONTAINING MARKER GENE
NURUL ASHIKrN MOHAMED ALl
This project is submitted in partial fulfillment of the requirements for the degree of Bachelor o f Science with Honours
(Resource Biotechnology)
Faculty o f Resource Science and Technology UNIVERSITY MALAYSIA SARAWA K
2005
CONTENTS
Content Page
Acknowledgement lV
Li st of Figure v
List ofTable Vl
Abstract Vll
Chapter 1 INTRODUCTION
11 Backgrollnd
111 Transfolmation
112 Restri ction enzyme
113 Polymerase Chain Reaction 2
114 Ligation 3
115 Plasmid 4
116 Reporter gene 9
117 Inducibl e promoter 10
118 Heat shock inducible promoter II
119 Tetracycline inducible promoter II
1110 Ethanol inducible promoter 12
12 Research Objectives 14
CONTENTS
Content Page
Chapter 2 MATERIAL AND METHOD
21 Preparation ofOvemight BacteIial Culture for Preparation of 15
Competent Cell s
22 CaCh bacteria l competent cells preparati on 15
23 Bacterial plasmid transformation 16
24 Mini-Prep Iso lation of Double Stranded Plasmid DNA from 16
Bacterial Culture
25 Restriction Enzyme Analysis 17
26 Agarose gel electrophoresis 18
27 Polymerase Chain reac tion 19
28 DNA extraction from agarose gel 2 1
29 Calf Intestine Alkaline Phosphate 21
210 Ligation 22
11
CO TENTS
Content Page
Chapter 3 RESULT AND DISCUSSION
3 1 Bacterial plasmid transfOlmation 23
32 Mini prep plasmid isolation 23
33 Restriction enzyme 24
34 Polymerase Chain Reaction 29
35 Ligation 3 1
Chapter 4 CONCLUS ION AND RECOMMENDATION 34
REFERENCES 35
APPENDIX 38
III
Acknowledgement
I would like to express my sincere app rec iation to my supervisor Dr Hairul
Azman Roslan for hi s guidance throughout this project A special thanks to my family
master students and my fe llow friends for their explanation and help during this project
IV
LIST OF FIGURE
Content Page
Figure I Restri ction maps ofpAGS 4
Figure 2 Restriction maps of pALS 5
Figure 3 Restriction maps ofpSRN 6
Figure 4 Restriction maps ofpGPTV 7
Figure 5 Graph expression of inducible gene JI
Figure 6 Mechanism of induc ible promoter 12
Figure 7 Plasmid iso lation 23
Figure 8 Restriction enzyme digestion 25
Figure 9 Restriction enzyme digest ofpAGS and pALS with HindIlI 26
Figure 10 Restriction enzyme digest pSRN and pGPTV with Hindlll 27
Figure 11 Result ofPCR to detennine the genotype of p lasmid 28
Figure 12 Plasmid isolation after digestion 29
Figure 13 Result of PCR 30
Figure 14 Possible orientation 31
v
LIST OF TABLE
Content Page
Table I List the reagent and volume of restriction enzyme ana lysis 16
Table 2 Lists the amount and volume used in PCR reaction 18
Table 3 List the reagent and vo lume used to prepared PCR reaction 19
using premix Qiagen PCR mixture
Table 4 PCR cycle used in routine determination of GUS LUC and 19
al cR gene in the plasmids
Table 5 The sequences of primers used for PCR work 20
Table 6 List the reagent and volume used to prepared PCR reaction usin g 2 1
premix Qiagen PCR mixtu re
Table 7 Lists the reagent and vo lume llsed for ligation process 21
Table 8 Shows the pl as mids restriction enzyme used to detect the insert 24
and the size of insert afteT digeslon
Table 9 Combination of pIimers used to detect the plasmid using PCR 28
Table 10 Combination ofprimern used to detec t the plasmid ll sing PCR 30
VI
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
CONTENTS
Content Page
Acknowledgement lV
Li st of Figure v
List ofTable Vl
Abstract Vll
Chapter 1 INTRODUCTION
11 Backgrollnd
111 Transfolmation
112 Restri ction enzyme
113 Polymerase Chain Reaction 2
114 Ligation 3
115 Plasmid 4
116 Reporter gene 9
117 Inducibl e promoter 10
118 Heat shock inducible promoter II
119 Tetracycline inducible promoter II
1110 Ethanol inducible promoter 12
12 Research Objectives 14
CONTENTS
Content Page
Chapter 2 MATERIAL AND METHOD
21 Preparation ofOvemight BacteIial Culture for Preparation of 15
Competent Cell s
22 CaCh bacteria l competent cells preparati on 15
23 Bacterial plasmid transformation 16
24 Mini-Prep Iso lation of Double Stranded Plasmid DNA from 16
Bacterial Culture
25 Restriction Enzyme Analysis 17
26 Agarose gel electrophoresis 18
27 Polymerase Chain reac tion 19
28 DNA extraction from agarose gel 2 1
29 Calf Intestine Alkaline Phosphate 21
210 Ligation 22
11
CO TENTS
Content Page
Chapter 3 RESULT AND DISCUSSION
3 1 Bacterial plasmid transfOlmation 23
32 Mini prep plasmid isolation 23
33 Restriction enzyme 24
34 Polymerase Chain Reaction 29
35 Ligation 3 1
Chapter 4 CONCLUS ION AND RECOMMENDATION 34
REFERENCES 35
APPENDIX 38
III
Acknowledgement
I would like to express my sincere app rec iation to my supervisor Dr Hairul
Azman Roslan for hi s guidance throughout this project A special thanks to my family
master students and my fe llow friends for their explanation and help during this project
IV
LIST OF FIGURE
Content Page
Figure I Restri ction maps ofpAGS 4
Figure 2 Restriction maps of pALS 5
Figure 3 Restriction maps ofpSRN 6
Figure 4 Restriction maps ofpGPTV 7
Figure 5 Graph expression of inducible gene JI
Figure 6 Mechanism of induc ible promoter 12
Figure 7 Plasmid iso lation 23
Figure 8 Restriction enzyme digestion 25
Figure 9 Restriction enzyme digest ofpAGS and pALS with HindIlI 26
Figure 10 Restriction enzyme digest pSRN and pGPTV with Hindlll 27
Figure 11 Result ofPCR to detennine the genotype of p lasmid 28
Figure 12 Plasmid isolation after digestion 29
Figure 13 Result of PCR 30
Figure 14 Possible orientation 31
v
LIST OF TABLE
Content Page
Table I List the reagent and volume of restriction enzyme ana lysis 16
Table 2 Lists the amount and volume used in PCR reaction 18
Table 3 List the reagent and vo lume used to prepared PCR reaction 19
using premix Qiagen PCR mixture
Table 4 PCR cycle used in routine determination of GUS LUC and 19
al cR gene in the plasmids
Table 5 The sequences of primers used for PCR work 20
Table 6 List the reagent and volume used to prepared PCR reaction usin g 2 1
premix Qiagen PCR mixtu re
Table 7 Lists the reagent and vo lume llsed for ligation process 21
Table 8 Shows the pl as mids restriction enzyme used to detect the insert 24
and the size of insert afteT digeslon
Table 9 Combination of pIimers used to detect the plasmid using PCR 28
Table 10 Combination ofprimern used to detec t the plasmid ll sing PCR 30
VI
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
CONTENTS
Content Page
Chapter 2 MATERIAL AND METHOD
21 Preparation ofOvemight BacteIial Culture for Preparation of 15
Competent Cell s
22 CaCh bacteria l competent cells preparati on 15
23 Bacterial plasmid transformation 16
24 Mini-Prep Iso lation of Double Stranded Plasmid DNA from 16
Bacterial Culture
25 Restriction Enzyme Analysis 17
26 Agarose gel electrophoresis 18
27 Polymerase Chain reac tion 19
28 DNA extraction from agarose gel 2 1
29 Calf Intestine Alkaline Phosphate 21
210 Ligation 22
11
CO TENTS
Content Page
Chapter 3 RESULT AND DISCUSSION
3 1 Bacterial plasmid transfOlmation 23
32 Mini prep plasmid isolation 23
33 Restriction enzyme 24
34 Polymerase Chain Reaction 29
35 Ligation 3 1
Chapter 4 CONCLUS ION AND RECOMMENDATION 34
REFERENCES 35
APPENDIX 38
III
Acknowledgement
I would like to express my sincere app rec iation to my supervisor Dr Hairul
Azman Roslan for hi s guidance throughout this project A special thanks to my family
master students and my fe llow friends for their explanation and help during this project
IV
LIST OF FIGURE
Content Page
Figure I Restri ction maps ofpAGS 4
Figure 2 Restriction maps of pALS 5
Figure 3 Restriction maps ofpSRN 6
Figure 4 Restriction maps ofpGPTV 7
Figure 5 Graph expression of inducible gene JI
Figure 6 Mechanism of induc ible promoter 12
Figure 7 Plasmid iso lation 23
Figure 8 Restriction enzyme digestion 25
Figure 9 Restriction enzyme digest ofpAGS and pALS with HindIlI 26
Figure 10 Restriction enzyme digest pSRN and pGPTV with Hindlll 27
Figure 11 Result ofPCR to detennine the genotype of p lasmid 28
Figure 12 Plasmid isolation after digestion 29
Figure 13 Result of PCR 30
Figure 14 Possible orientation 31
v
LIST OF TABLE
Content Page
Table I List the reagent and volume of restriction enzyme ana lysis 16
Table 2 Lists the amount and volume used in PCR reaction 18
Table 3 List the reagent and vo lume used to prepared PCR reaction 19
using premix Qiagen PCR mixture
Table 4 PCR cycle used in routine determination of GUS LUC and 19
al cR gene in the plasmids
Table 5 The sequences of primers used for PCR work 20
Table 6 List the reagent and volume used to prepared PCR reaction usin g 2 1
premix Qiagen PCR mixtu re
Table 7 Lists the reagent and vo lume llsed for ligation process 21
Table 8 Shows the pl as mids restriction enzyme used to detect the insert 24
and the size of insert afteT digeslon
Table 9 Combination of pIimers used to detect the plasmid using PCR 28
Table 10 Combination ofprimern used to detec t the plasmid ll sing PCR 30
VI
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
CO TENTS
Content Page
Chapter 3 RESULT AND DISCUSSION
3 1 Bacterial plasmid transfOlmation 23
32 Mini prep plasmid isolation 23
33 Restriction enzyme 24
34 Polymerase Chain Reaction 29
35 Ligation 3 1
Chapter 4 CONCLUS ION AND RECOMMENDATION 34
REFERENCES 35
APPENDIX 38
III
Acknowledgement
I would like to express my sincere app rec iation to my supervisor Dr Hairul
Azman Roslan for hi s guidance throughout this project A special thanks to my family
master students and my fe llow friends for their explanation and help during this project
IV
LIST OF FIGURE
Content Page
Figure I Restri ction maps ofpAGS 4
Figure 2 Restriction maps of pALS 5
Figure 3 Restriction maps ofpSRN 6
Figure 4 Restriction maps ofpGPTV 7
Figure 5 Graph expression of inducible gene JI
Figure 6 Mechanism of induc ible promoter 12
Figure 7 Plasmid iso lation 23
Figure 8 Restriction enzyme digestion 25
Figure 9 Restriction enzyme digest ofpAGS and pALS with HindIlI 26
Figure 10 Restriction enzyme digest pSRN and pGPTV with Hindlll 27
Figure 11 Result ofPCR to detennine the genotype of p lasmid 28
Figure 12 Plasmid isolation after digestion 29
Figure 13 Result of PCR 30
Figure 14 Possible orientation 31
v
LIST OF TABLE
Content Page
Table I List the reagent and volume of restriction enzyme ana lysis 16
Table 2 Lists the amount and volume used in PCR reaction 18
Table 3 List the reagent and vo lume used to prepared PCR reaction 19
using premix Qiagen PCR mixture
Table 4 PCR cycle used in routine determination of GUS LUC and 19
al cR gene in the plasmids
Table 5 The sequences of primers used for PCR work 20
Table 6 List the reagent and volume used to prepared PCR reaction usin g 2 1
premix Qiagen PCR mixtu re
Table 7 Lists the reagent and vo lume llsed for ligation process 21
Table 8 Shows the pl as mids restriction enzyme used to detect the insert 24
and the size of insert afteT digeslon
Table 9 Combination of pIimers used to detect the plasmid using PCR 28
Table 10 Combination ofprimern used to detec t the plasmid ll sing PCR 30
VI
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
Acknowledgement
I would like to express my sincere app rec iation to my supervisor Dr Hairul
Azman Roslan for hi s guidance throughout this project A special thanks to my family
master students and my fe llow friends for their explanation and help during this project
IV
LIST OF FIGURE
Content Page
Figure I Restri ction maps ofpAGS 4
Figure 2 Restriction maps of pALS 5
Figure 3 Restriction maps ofpSRN 6
Figure 4 Restriction maps ofpGPTV 7
Figure 5 Graph expression of inducible gene JI
Figure 6 Mechanism of induc ible promoter 12
Figure 7 Plasmid iso lation 23
Figure 8 Restriction enzyme digestion 25
Figure 9 Restriction enzyme digest ofpAGS and pALS with HindIlI 26
Figure 10 Restriction enzyme digest pSRN and pGPTV with Hindlll 27
Figure 11 Result ofPCR to detennine the genotype of p lasmid 28
Figure 12 Plasmid isolation after digestion 29
Figure 13 Result of PCR 30
Figure 14 Possible orientation 31
v
LIST OF TABLE
Content Page
Table I List the reagent and volume of restriction enzyme ana lysis 16
Table 2 Lists the amount and volume used in PCR reaction 18
Table 3 List the reagent and vo lume used to prepared PCR reaction 19
using premix Qiagen PCR mixture
Table 4 PCR cycle used in routine determination of GUS LUC and 19
al cR gene in the plasmids
Table 5 The sequences of primers used for PCR work 20
Table 6 List the reagent and volume used to prepared PCR reaction usin g 2 1
premix Qiagen PCR mixtu re
Table 7 Lists the reagent and vo lume llsed for ligation process 21
Table 8 Shows the pl as mids restriction enzyme used to detect the insert 24
and the size of insert afteT digeslon
Table 9 Combination of pIimers used to detect the plasmid using PCR 28
Table 10 Combination ofprimern used to detec t the plasmid ll sing PCR 30
VI
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
LIST OF FIGURE
Content Page
Figure I Restri ction maps ofpAGS 4
Figure 2 Restriction maps of pALS 5
Figure 3 Restriction maps ofpSRN 6
Figure 4 Restriction maps ofpGPTV 7
Figure 5 Graph expression of inducible gene JI
Figure 6 Mechanism of induc ible promoter 12
Figure 7 Plasmid iso lation 23
Figure 8 Restriction enzyme digestion 25
Figure 9 Restriction enzyme digest ofpAGS and pALS with HindIlI 26
Figure 10 Restriction enzyme digest pSRN and pGPTV with Hindlll 27
Figure 11 Result ofPCR to detennine the genotype of p lasmid 28
Figure 12 Plasmid isolation after digestion 29
Figure 13 Result of PCR 30
Figure 14 Possible orientation 31
v
LIST OF TABLE
Content Page
Table I List the reagent and volume of restriction enzyme ana lysis 16
Table 2 Lists the amount and volume used in PCR reaction 18
Table 3 List the reagent and vo lume used to prepared PCR reaction 19
using premix Qiagen PCR mixture
Table 4 PCR cycle used in routine determination of GUS LUC and 19
al cR gene in the plasmids
Table 5 The sequences of primers used for PCR work 20
Table 6 List the reagent and volume used to prepared PCR reaction usin g 2 1
premix Qiagen PCR mixtu re
Table 7 Lists the reagent and vo lume llsed for ligation process 21
Table 8 Shows the pl as mids restriction enzyme used to detect the insert 24
and the size of insert afteT digeslon
Table 9 Combination of pIimers used to detect the plasmid using PCR 28
Table 10 Combination ofprimern used to detec t the plasmid ll sing PCR 30
VI
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
LIST OF TABLE
Content Page
Table I List the reagent and volume of restriction enzyme ana lysis 16
Table 2 Lists the amount and volume used in PCR reaction 18
Table 3 List the reagent and vo lume used to prepared PCR reaction 19
using premix Qiagen PCR mixture
Table 4 PCR cycle used in routine determination of GUS LUC and 19
al cR gene in the plasmids
Table 5 The sequences of primers used for PCR work 20
Table 6 List the reagent and volume used to prepared PCR reaction usin g 2 1
premix Qiagen PCR mixtu re
Table 7 Lists the reagent and vo lume llsed for ligation process 21
Table 8 Shows the pl as mids restriction enzyme used to detect the insert 24
and the size of insert afteT digeslon
Table 9 Combination of pIimers used to detect the plasmid using PCR 28
Table 10 Combination ofprimern used to detec t the plasmid ll sing PCR 30
VI
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
Construction of An Inducible Vector Containing Marker Gene
Nurul Ashikin bt Mohamed Ali
Resource Biotechn ology Faculty of resource Science and Technology
University Malaysia Sarawak (UNIMAS)
ABSTRACT
This study concerns on understanding the function of inducible promoter that can controlled the expression in plant The used of controllable promoter region permit a regulated of a foreign gene in plant The cassette contain the induce promoter carry a maker gene (GUS and LUC) was constructed in pUC plasmid This construct based on the alcR regulatOlY system from the fungus Aspergilus nidulans with the application of ethanol as inducer
Keywords Plant expression system ethanol ALCR LUCIGUS
ABSTRAK
Kajian ini adalah untuk memahami fimgsi pengalak promoter yang menga IVa I pengekspresan dalam tumbuhan Penggunaan pengawalan promoter menyebabkan regu las gen asing dalam tumbuhan Casselle yang mengandung promoter penggalak dan mengandungi gen pemtnjuk (GUS dan LUC) ini dibina di dalam plasmid pUc Pembinaan ini berdasarkan sistem regulasi alcR yang berasal dari fimg i Aspergilus nidulans bersama aplikasi ethanol sebaga pengalak
Kata kunci Sistem pengekspresan tumbuhan ethanol ALCR LUCGUS
Vll
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
CHAPTER 1
INRODUCTION
11 Background
111 Transforma tion
The success of many molecular hiology protocols is directly related to the ability to
achieve efficient uptake of vector containing the DNA of interest and the most common
method ofbactelial transformation utilizes high level of calcium c101ide that facilitate the
entry of plasmid DNA vectors into E Coli by cousing structural alterations in the
bacterial cell wall (Becker el ai 1990)
There are three steps to introduce plasmid DNA into cel ls First step is preparation
of competent cells second step is transformation of competent cells and third step IS
se lection of transfOlmants (Becker et al 1990)
The factors influencing this efficiency are often related to conditions that render
cells competent or able to take up DNA (Becker el al 1990)
112Restriction enzyme
Restriction enzymes are endonucleases that cleave DNA in respon se to a
recognition site on the DNA (Ken drew e ai1994) Restriction endonucleases are
enzyme that recognize a specific base sequence of DNA Us ually 4 to 6 base pairs (bp)
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
long and then cleave the DNA at a defined position in relation to the sequences
Restriction endonucleases mean that of the completed digeation 0 f a partic ul ar sequences
of DNA by a specific or combination of enzyme will result in the production of a
reproducible set of fragment generate according to the frequency and the location of the
specific enzyme recognition sequence Plasmid DNA molecules can compare by
exchanging the number and size of fragment generated by the digestion of the DNA with
restriction endonucleases
113 Polymerase Chain Reaction (PCR)
PCR is a technique for the amplitying of spec ific nucleic acid sequences The
amplification IS achieved with a thermostable DNA polymerase synthetic
oligonucleotide primer and fo ur standard of deoxyribonucleo tide triphosphates (Towner
and Cockayne 1993)
Process of the PCR is divided into three source of actions are repeated in cycles
The first step is the denature of the duplex sample of the DNA following with the second
step is the annealing of the two primer to the opposite DNA strand and lastly step three is
the extension of the polymerase that is mediated with nucleotide addition to produce two
copies of dIe original sequence it is a panicular reaction that is controlled by
oligonucleo tide DNA strands Within a few hours millions of copies are produced The
primer for the PCR will on ly react with the specific binding site The process of the PCR
2
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
very much depends to the Thermus aquatiqus or also known as Taq polymerase The
major benefit of peR is that small amount of DNA is needed (Russell et aI 1992)
114 Ligation
Ligation is the process whereby a 5-3 phosphodiester bond is fonned between two
DNA fragment and usually mediated by DNA ligase (Kahl et al 2001) Before the
ligation process the plasmid taken from after the ge l extraction process be treated with
Calf Intestine Alkaline Phosphate (ClAP) Alkaline phosphate is an enzyme catalyzing of
the 5 terminal phosphate group from linear DNA It is used to prevent the
recircularizationand dimerization of plasmid vector DNA that has been cleaved with an
endonuclease (Kah I et al 200 I) Phosphatase treatment thus increases the probability of
fonnation of recombinant molecules since circularization of plasmid can only occur by
the insertion of non-phosphatase treated foreign DNA with a 5 tenninal phosphate at
each end
3
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
115 Plasmid
The plasmid that contained of a cell normally comptise less than 5 of the plasmid
profiles (Towner and Cockayne 1993) There are four types of plasmid used in this
research pAGS pALS pSRN and pGPTV
pAGS and pALS are derived from the same pUC 18 plasmid (Figure I and figure 2)
The vector genes are inserted between alcA and 3SSt (Roslan et aI 2001) pSRN was
from pUCIS (Figure 3) The GUSCaMV3SSalcRnos are insetted in EcoRl and
Hindlll site (Roslan et aI 200 I) pGPTV is a plant transformation vector that resistant in
agromyc in (Figure 4)
4
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
PSII Hi1ldUO
8(1mHI
pAGS
Hindlf
I
Xbal
BamH EcaR235
Figure I Restri ction map of plasmid pAGS that contain the aicA GUS gene and 35St
5
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
-
---
-- ----------
jESI I) II
Hld )fIHll uflf X(1 i j
l ( BClIII H
pALS
ttlA I J
l i
-5
fcoRV 1
XlO t j Vml
Srf
(u l
SSI
-- Sma
E(U I
Xbal
rII1el1
Figure 2 Restriction map ofplasmid pALS that contain the alcA LUC gene and 35St
6
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
- ---
-- --- ------ ---
EcoRI Spe l --
J No l I
Avril ~===L ___ --~- _ I
pSRN4
Fse l
625 Kb
oi
Kplll
BamH
Apet
Ishy
Eugl BamHI Xbul Sail
Figure 3 Restriction map of plasmid pSRN that contain the alcR CaMV35S and nos
7
- -- ~
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
pGPTV- HPT (1 3950 bpI
Figure 4 Restriction map ofplasmid pGPTV-hpt
8
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
116 Reporter gene
Reporter enzymes often are used to monitor gene expression Several reporter gene
systems have been developed which differ in ease of use cost sensitivity versatility and
safety
The p-glucuronidase (GUS) enzyme from E coli (EC32131) has been well
documented to provide desirable characteristics as a maker gene in performed plants
GUS reporter gene system has many advantages including stable expression of E coli
GUS enzyme no intelference with normal plant metabolism and low intrinsic GUS
activity in higher plant The GUS gene is usually used in a gene fusion (Bains 1993)
This means that the GUS coding sequence is under the direction of the controlling
sequence of another gene For this exercise the GUS gene is under the control of the
Cauliflower Mosaic Virus 35S promotor
The luciferase (LUC) enzyme from the North American firefly Pho tinus pyrais
extensively as a reporter in studies of plant gene expression (Kahl 200 I) Luciferase
produces a characteristic fluorescence Fluorescence indicates successful integration of
the transgene into a particular organism
9
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
117 Inducible Promoter
Expression of DNA sequences coding for protein is important process in plant
All organism have switches known as promoter which direct the expression of gene at
defined times in defined cells Some continuously produce their product in all cells but
many gene are regulated in response to cell differentiation development or some extemal
stimulus or inducible promoter
In plant the most commonly used expression system employs the 35S promoter
fium the Cau liflower Mosaic Virus (CaMV35S) that give high level of expression in
most cell types and plant species (Veylder et aI 2000) Controllable promoter permits
very precise wi th the optimal level of expression and time at which the DNA sequences is
expressed Controllable promoter is controlled by an inducer (VeyJder et aI 2000)
For a good inducible system expression should be linked only to the presence of
the inducing compound and the inducing compound should not intertere with normal
plant development nor be toxic to the organism (Veylder et aI 2000) Inducible system
can be group into two The first utili zes sequences of plant origin for example heat shock
inducible promoter The second gro up is a chemical inducible system based on non plant
system (Veylder et af 2000) tor example group of copper and ethanoL This has great
benefits for both research and agriculture using GM plants
Sequence into the target cells can be accomplished by variety of techniques such
as eiectroporation microinjection agrobacterium in fect ion and liposome or
microprojectile trans formation
[0
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
118 Hea t shock inducible promoter
Heat shock as a chemical inducible system had been used in several expelimentor
research expecially for research in soybean Arabidopsis and some tobacco system for
studies of expression in transgenic plants A soybean heat shock promoter will be used to
dri ve the inducible expression of evFp-LexA and Gal-ECFD
The advantage of used heat shock promoter system level ofthe induction with thi s
method move precisely by simply changing the time of incubation at the higher
temperature But the application is limited to the laboratory as it would not be feasible to
apply a controlled heat shock treatment in the field (Roslan et al 200 I)
119 Tetracycline inducible promoter
Tetracyclline is an example of antibiotic inducible promoter A preferred system
is the Tn 10 tet repressor system which is respond site to tetracycline In this system a
modified Cauliflower Mosaic VilUS (CaMV) 35S promoter contai ning one or more
preferably three tet operon is used (Veylder et ai 2000) the Tn 10 tet repressor gene
produces a repressor protein that bind to the tet operon and prevents the expression of the
gene to which the promoter is linked The presence oftetracyciine inhibits binding of the
Tn 10 tet repressor to the tet operon allowing fiee expression of the linked gene This
11
----shy
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
system is preferred because the stimulus tetracycl ine is not one to which the plant world
normall y be exposed so its application can be contro lled
Tetracycline also has no harmfill effects on plants ar animal not impede the
nonnal development of the plant and the residual amount left on the seed or p lant have no
sign ifican t environmental impac t( Veylder et ai 2000)
1110 Ethanol inducible promoter
In thi s research we use a system based on the alc regulatory system in the fungus
Aspergillus nidulans
The transcription factor (ALCR) activates expression of the aleA promoter in the
presence of ethanol The used of ethanol as inducible promoter is an advantage because is
of ethanol is inexpensive nontoxic and application can be full y control (figure 1)
(Roslan ef al 200 1) It is also biodegradable and may be safel y applied to the roots and
leaves ofplants in the field
ALCR is specific activator of the Aspergilus nidulans ethano l utilization
mediating the induction of its own transcription and that of structural genes aleA and
aled encoding for alcoho l dehydrogenase and aldehyde deydrogenase respectively
(Shafqaf et al 1998 )
12
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
HJlJU
c 2ltgtn()Q
~ -noo ~
c I SOl) c
~ c IOOU 1
50 0
0
middotmiddotth ln(1
ldtlLIGtl
- 1 (I 0 2
Huungt
Figure 5 Graph shown the expression of an inducible gene before and after the addition of
ethanol
e I
reporter
Figure 6 Shows the mechanism reaction ethanol as inducer
J3
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
The alcA and alcR inducible promoter system is a two component system
invo lving DNA sequences coding for the alcA promoter and the alcR protein The alcR
gene encodes the 861 amino ac id ALCR protein ( Felenbock el la 1988) ALCR protein
acti vates the alcA promoter in the presence of inducer alcR is important it preferabl y be a
tissue or organ se lective promoter such as a leaf fru it grain endospelm on seed and when
the method of the presence invention is for used in cerial crops the expression of alcR is
disirably controlled by a seed promoter and for for grain the expression of alcR involved
in starch synthesis
In this research the alcR is being continously expressed by CaMV35S promoter
and by contain the nos terminator (Bevan el ai 1983) alcA promoter is fused to the
TATA box of the CaMV35S promoter (Roslan e ai 200 ) The TATA box asists in
directing RNA polymerase II to the initiation site downstream on DNA RNA polymerase
cannot recognized to the TAT A box on its self until the transc ription factor (ALCR) bind
to the T AT A box then the transcription begins
13 Research objective
There are two main objectives in thi s research first to check the plasmid ]JAGS
pALS pSRN and pGPTV from stock collection and second is to create a cassette
containing an inducible promoter call)ing a m arker gene
14
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
CHAPTER 2
MATERIAL AND METHOD
21 Preparation of Overnight Bacterial Culture for Preparation of Competent Cells
Single co lony from the agar plate of E coli was inocu lated into bijou bottle
containing 5 ml of LB media The media is in a shaking incubator at 37degC The culture is
kept overnight at 4degC for use in the CaCh bacterial competen t cell preparation
22 CaCh bacterial competent cells preparation
10 ml of overnight cell was transferred into a 60 ml of Luria Broth The cell was
vigorous shaking to provide proper aeration in 37degC to a density of 5 X 109 cellsml (OD
590=04) of approximately After the cells have reached an appropriate density the ce lls
are removed and pipeted into a prechilled steri le centrifuge tube and put on ice for 10
minute The cells were then washed by gently resuspending them in 25 ml iced-cold
100mM CaCh The cells were then kept on ice for 10 minutes and recentrifuge After the
centrifuge process the supematant was decanted the cell before resuspended in 25 ml of
cold sterile 100mM CaCh and further incubated on ice for I hour before used or stored
in - 70degC
15
