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CLONING, TRANSFORMATION AND EXPRESSION
OF cDNA OF HUMAN scFv AGAINST
HEPATOCELLULAR CARCINOMA IN INDICA RICE
VARIETY (IR-64)
A THESIS
Submitted by
AADARSH RS
for the award of the degree
of
DOCTOR OF PHILOSOPHY
DEPARTMENT OF INDUSTRIAL BIOTECHNOLOGY
Dr.M.G.R
EDUCATIONAL AND RESEARCH INSTITUTE
UNIVERSITY
(Declared u/s 3 of UGC Act.1956)
CHENNAI 600 095
JUNE 2011
ii
BONAFIDE CERTIFICATE
I certify that this thesis entitled “CLONING, TRANSFORMATION AND
EXPRESSION OF cDNA OF HUMAN scFv AGAINST HEPATOCELLULAR
CARCINOMA IN INDICA RICE VARIETY (IR-64)” is the bonafide work of
Mrs. AADARSH R S M.Sc., who carried out the research under my supervision.
Certified further, that to the best of my knowledge the work reported herein does not
form part of any other thesis or dissertation of the basis of which a degree or award
was conferred on an earlier occasion on this or any other candidate.
Dr. P. BALAKRISHNA MURTHY Ph.D., PDF, D.Sc.
Director-IIBAT
(Supervisor)
Dr. M. DEECARAMAN Ph.D., PDF
Dean of Industrial Biotechnology
Dr. M.G.R Educational and Research Institute
(Co- Supervisor)
iii
AADARSH R S, M. Sc., Department of Biotechnology,
Research Scholar IIBAT,
Padappai – 601 301.
DECLARATION
This is to certify that the thesis titled, “CLONING, TRANSFORMATION AND
EXPRESSION OF cDNA OF HUMAN scFv AGAINST HEPATOCELLULAR
CARCINOMA IN INDICA RICE VARIETY (IR-64)” submitted by me to the Dr.
M.G.R. Educational and Research Institute University for the award of the degree of
Doctor of Philosophy is a Bonafide record of research work carried out by me under
the supervision of Dr. P. BALAKRISHNA MURTHY, Director, IIBAT and Dr. M.
DEECARAMAN, Dean, Department of Industrial Biotechnology, Dr. M.G.R
Educational and Research Institute, during the period from 2007 to 2011. The contents
of this thesis, in full or in parts, have not been submitted to any other Institute or
University for the award of any degree or diploma.
AADARSH RS
iv
ABSTRACT
v
ACKNOWLEGMENT
I thank the Almighty God, who has helped me to take another step in my life.
In every step of my life He has been and will be my rock, and my fortress, and my
deliverer, my rock, in whom I take refuge, my shield, and the horn of my salvation, my
stronghold.
My sincere respect and thanks to our Chancellor Thiru A.C. Shanmugan
B.A., B.L., and Pro-Chancellor Thiru A.C.S. Arunkumar, Dr. M.G.R. Educational and
Research Institute for providing me an opportunity to do research work. I also thank
Dr.P. Aravindan, Vice Chancellor and Dr. P.T. Manogaran, Vice President
(Academic) for sound advice and guidance.
It is difficult to overstate my sincere gratitude to my Ph.D. supervisor, Dr. P.
Balakrishna Murthy, Ph.D., PDF, D.Sc, Director, IIBAT, for providing facility and
support to carry out my research work. With his enthusiasm, his inspiration, and his
great efforts to explain things clearly and simply, he helped to make research work
exciting for me. Throughout my research period, he provided encouragement, sound
advice, good teaching, good company, and lots of good ideas. I would have been lost
without him.
vi
I warmly thank my Co-Supervisor, Dr. M. Deecaraman Ph.D., PDF, Dean
of Industrial Biotechnology, Dr. M.G.R Educational and Research Institute for his
valuable advice and friendly help. His extensive discussions around my work and
interesting explorations on my research work have been very helpful.
I am deeply grateful to Dr. R. Shridar, Head of the Department of
Biotechnology, IIBAT, for his detailed and constructive comments, and for his
important support throughout my research work.
I wish to express my warm and sincere thanks to Dr. Osmat Azzam
Jefferson (CAMBIA, Australia) for providing me with plasmid pCAMBIA 1301 for
cloning.
I am thankful to Dr. Tomasz Pniewski, (Institute of Plant Genetics, Polish
Academy of Sciences, Poland) for providing me Agrobacterium tumefaciens EHA105
strain for transformation.
I am thankful to Dr. K. Veluthambi (Department of plant genetics, MKU
University, India) for providing me with E. coli DH5α strain for cloning and for his
extensive suggestion about my research work, which have been very helpful.
I express my sincere thanks to Dr. A. Ramesh, Head of the Department of
Analytical chemistry, IIBAT, for his constant encouragement and support.
I wish to thank Dr. Rama Vaidyanathan and Dr. M. Vijaya Lakshmi, Head
of Industrial Biotechnology, Dr. M.G.R Educational and Research Institute for the
support and encouragement.
vii
I wish to thank my friends and colleagues, especially Mrs. Deepa
Surendirababu and Mrs. Sathya Diwakar, for helping me get through the difficult
times, and for all the emotional support, entertainment, and caring they provided.
I greatly express my sincere gratitude for co-operation and valuable support
from my beloved husband G.K. Prasanna, my sweet daughter K.P. Shreenidhi, my
father A. Radhakrishnan, my mother D. Sujatha and other family members without
whom I would not have achieved anything.
AADARSH R S
viii
TABLE OF CONTENTS
CHAPTERS TITLE
PAGE
NO
ABSTRACT iv
LIST OF TABLES xii
LIST OF FIGURES xiv
LIST OF SYMBOLS & ABBREVIATIONS Xix
1. INTRODUCTION 1
2. LITERATURE REVIEW 4
3. AIM AND OBJECTIVE OF THE WORK 31
4. MATERIALS AND METHODS 34
4.1. GENE CLONING USING VECTOR NTI SOFTWARE 35
4.2. SYNTHESIS OF HCC-scFv GENE-GENEART 40
ix
4.3. 4.3. TRANSFORMATION OF PLASMID pCAMBIA 1301 AND
pMA-T INTO E. coli DH5α AND SCREENING OF
TRANSFORMED E. coli DH5α STRAIN
41
4.4. ISOLATION OF PLASMID pCAMBIA 1301 AND pMA-T
from TRANSFORMED E. coli DH5α AND CONFIRMATION
BY GEL ELECTROPHORESIS
47
4.5. CLONING OF HCC-scFv FROM pMA-T PLASMID INTO
pCAMBIA 301
56
4.5.1. RESTRICTION DIGESTION OF pCAMBIA 1301 AND
pMA-T for CLONING
57
4.5.2. LIGATION OF HCC-scFv AND pCAMBIA 1301 61
4.6. TRANSFORMATION OF pCAMBIA 1301-scFv FROM E. coli
DH5α INTO Agrobacterium tumefaciens EHA105
70
4.7. CALLUS INDUCTION OF IR64 RICE VARIETY FOR
TRANSFORMATION
76
4.8. TRANSFORMATION OF pCAMBIA 1301-HCC-scFv FROM
Agrobacterium tumefaciens EHA 105 INTO CALLUS BY
Agrobacterium- MEDIATED TRANSFORMATION
80
4.9. EXTRACTION OF TOTAL GENOMIC DNA FROM PLANT
TISSUES FOR MOLECULAR ANALYSIS
90
x
4.10. MOLECULAR ANALYSIS OF PUTATIVE TRANSFORMED
PLANTS BY POLYMERASE CHAIN REACTION (PCR)
97
5. RESULTS AND DISCUSSIONS 103
5.1. GENE CLONING USING VECTOR NTI SOFTWARE 104
5.2. SYNTHESIS OF HCC-scFv GENE-GENEART 122
5.3. TRANSFORMATION OF PLASMID pCAMBIA 1301 AND
pMA-T INTO E. coli DH5α AND SCREENING OF
TRANSFORMED E. coli DH5α STRAIN
134
5.4. ISOLATION OF PLASMID pCAMBIA 1301 AND pMA-T
from TRANSFORMED E. coli DH5α AND CONFIRMATION
BY GEL ELECTROPHORESIS
141
5.5. CLONING OF HCC-scFv FROM pMA-T PLASMID INTO
pCAMBIA 301
144
5.5.1. RESTRICTION DIGESTION OF pCAMBIA 1301 AND
pMA-T for CLONING
144
5.5.2. LIGATION OF HCC-scFv AND pCAMBIA 1301
146
5.6. TRANSFORMATION OF pCAMBIA 1301-scFv FROM E. coli
DH5α INTO Agrobacterium tumefaciens EHA105 149
xi
5.7. CALLUS INDUCTION OF IR64 RICE VARIETY FOR
TRANSFORMATION
152
5.8. TRANSFORMATION OF pCAMBIA 1301-HCC-scFv FROM
Agrobacterium tumefaciens EHA 105 INTO CALLUS BY
Agrobacterium- MEDIATED TRANSFORMATION
154
5.9. EXTRACTION OF TOTAL GENOMIC DNA FROM PLANT
TISSUES FOR MOLECULAR ANALYSIS
156
5.10. MOLECULAR ANALYSIS OF PUTATIVE
TRANSFORMED PLANTS BY POLYMERASE CHAIN
REACTION (PCR)
157
6. SUMMARY 159
7. CONCLUSION AND SCOPE OF FUTURE WORK 162
APPENDICES 1-3
REFERENCES
LIST OF PUBLICATIONS
xii
LIST OF TABLES
TABLE
No
TITLE
PAGE
No
Table
2.5.1
Plant expression systems used for recombinant protein
production
10
Table
2.6.8.1
Antibodies expressed in plant expression system 26-27
Table
4.5.1.1
Restriction Digestion set up: pCAMBIA 1301: Vector
DNA 59
Table
4.5.1.2
Restriction Digestion set up: pMA-T (HCC-scFv): Insert
DNA 59
Table
4.5.2.1
Setup of Ligation reaction 66
Table
4.5.2.2
Controls for Ligation setup. 67
Table
4.5.2.3
Controls kept for the cloning experiment. 59
xiii
Table
4.7.1
Chemical composition of MS 2, 4-D medium 78
Table
4.8.1
Chemical composition of AAM Medium 84
Table
4.8.2
Chemical composition of N6 Medium 85
Table
4.10.1
Reaction mixture for PCR setup 101
Table
5.3.1
Screening of the transformed E. coli containing
pCAMBIA 1301 on LB medium containing kanamycin. 135
Table
5.3.2
Screening of the transformed E. coli containing
pCAMBIA 1301 on LB medium containing -
kanamycin+X-gal +IPTG.
137
Table
5.3.3
Screening of the transformed E. coli containing pMA-T-
HCC-scFv on LB medium containing ampicillin. 139
xiv
LIST OF FIGURES
FIGURE No TITLE PAGE
No
Figure 2.6.2.1
Schematic representation of full length antibody and
partial antibody used for therapeutic antibody
production
12
Figure 2.6.6.1
N-glycans structure in plants and mammals. 19
Figure 2.6.7.1
Glycoengineering/ Humanization of plantibodies 21
Figure 4.5.1
Illustration of cloning HCC-scFv gene into pCAMBIA
1301.
56
Figure 4.6.1
Illustration of transformation of pCAMBIA 1301-
HCC-scFv into A.tumefaciens EHA 105 by triparental
mating
71
Figure 4.8.1
Illustration of transformation of pCAMBIA 1301-
HCC-scFv into IR 64.
81
xv
Figure 5.1.1
Analysis of Gene sequence of HCC-scFv: AY686498
downloaded from the Gen Bank using vector NTI
software
106
Figure 5.1.2
Analysis of Gene Map of HCC-scFv: AY686498
downloaded from the Gen Bank using vector NTI
software
106
Figure 5.1.3
Modified Gene sequence of HCC-scFv: AY686498
downloaded from the Gen Bank using vector NTI
software
107
Figure 5.1.4
Modified Gene Map of HCC-scFv: AY686498
downloaded from the Gen Bank using vector NTI
software
107
Figure 5.1.5
Analysis of Gene sequence of pCAMBIA 1301
downloaded from the Gen Bank using vector NTI
software
113
Figure 5.1.6
Analysis of Gene Map of pCAMBIA 1301 downloaded
from the Gen Bank using vector NTI software
114
Figure 5.1.7
Gene Map: Cloning of pCAMBIA 1301 and HCC-scFv
to form pCAMBIA 1301-HCC-scFv using vector NTI
software
115
xvi
Figure 5.1.8
Gene Sequence: Cloning of pCAMBIA 1301 and HCC-
scFv to form pCAMBIA 1301-HCC-scFv using vector
NTI software
121
Figure 5.2.1
Plot showing G+C content 123
Figure 5.2.3
Gene sequenced from the synthesized gene. 129
`
Figure 5.2.4
Sequence identity of the synthesized gene sequence
and the given sequence.
131
Figure 5.2.5
Gene sequence of HCC-scFv gene cloned into plasmid
pMA-T (pMA-T-HCC-scFv) with NcoI and PmlI
restriction site.
132
Figure 5.2.6
Gene Map of pMA-T containing HCC-scFv with
various restriction sites.
133
Figure 5.3.1
Transformation of pCAMBIA1301 into E. coli DH5α
and screening of the transformed cells on LB medium
containing Kanamycin antibiotic.
136
Figure 5.3.2
Screening of the E. coli DH5α transformed with
pCAMBIA1301 on LB medium containing
Kanamycin, X-gal and IPTG.
138
Figure 5.3.3
Transformation of pMA-T-HCC-scFv into E. coli 140
xvii
DH5α and screening of the transformed cells on LB
medium containing ampicillin antibiotic.
Figure 5.4.1
Plasmid isolated from transformed E. coli DH5α
containing pCAMBIA 1301.
142
Figure 5.4.2
Plasmid isolated from transformed E. coli DH5α
(pCAMBIA 1301 and pMA-T-HCC-scFv).
143
Figure 5.5.1
Restriction digestion of pCAMBIA 1301 and pMA-T
(HCC-scFv) with NcoI and PmlI restriction enzymes.
145
Figure 5.5.2.1
Transformation of the ligated pCAMBIA 1301 and
pMA-T (HCC-scFv) into E. coli DH5α and screened on
LB medium containing Kanamycin.
147
Figure 5.5.2.2
Confirmation of the transformed E. coli DH5α
containing ligated pCAMBIA 1301 and pMA-T (HCC-
scFv)
148
Figure 5.6.1 Transformation of pCAMBIA 1301-HCC-scFv into
A.tumefaciens EHA 105 by triparental mating and
screening on AB minimal medium containing
Kanamycin antibiotic
150
Figure 5.6.2
Transformation of pCAMBIA 1301-HCC-scFv into
A.tumefaciens EHA 105 by triparental mating. 151
Figure 5.7.1
Callus induction of IR4 using 2, 4 D-MS medium 153
xviii
Figure 5.8.1 Transformation of pCAMBIA 1301-HCC-scFv from
A.tumefaciens EHA 105 into IR64 and selection of the
transformed callus on Hygromycin.
155
Figure 5.10.1
Molecular analysis of the transformed plant (CaMV
35 S) by polymerase chain reaction
158
xix
LIST OF SYMBOLS & ABBREVIATIONS
µg/ml- micro gram per millilitre
2,4D - Dichlorophenoxy acetic acid
BiP- Binding protein
bp- Base pair
CaMV35S - Cauliflower mosaic virus 35S promoter
cDNA- Complementary Deoxyribonucleic acid
CTAB - Cetyltrimethylammonium bromide
DMF - dimethyl formamide
DNA - Deoxyribonucleic acid
EDTA - Ethylenediaminetetraacetic acid
ER- Endoplasmic reticulum
F (ab)’ 2 – Bivalent antigen binding fragment
Fab- Antigen binding fragment
Fc-Fragment constant
Fv- Fragment variable
g/L- gram per litre
g/mol- grams per mole
GRAS- Generally regarded as safe
HAMA- Human anti mouse antibody
HBV- Hepatitis B virus
HCC- Hepatocellular carcinoma
HCV- Hepatitis C virus
xx
HDEL - His-Asp-Glu-Leu (ER retention signal)
HIV- Human immunodeficiency virus
HSV- Herpes-simplex virus
IgA- Immunoglobulin A
IgD - Immunoglobulin D
IgE- Immunoglobulin E
IgG- Immunoglobulin G
IgM- Immunoglobulin M
IPTG (Iso propyl thiogalactoside or isopropyl beta-D-thiogalactopyranoside
Kb –kilo base pairs
KDEL - Lys-Asp-Glu-Leu (ER retention signal)
Kg/ha- kilogram per hectare
mg/Kg – milligram per kilogram
mg/L –milligram per litre
mg/ml- milligram per millilitre.
mM- millimolar
NAA- Naphthalene acetic acid
ng – nanogram
nm- nanometers
PSV- Protein storage vacuole
PVP - Polyvinylpyrrolidone
RNAi - Ribonucleic acid interference
scFv- Single chain variable fragment
SDS- sodium dodecyl sulfate
SEKDEL - Ser-Glu-Lys-Asp-Glu-Leu (ER retention signal)
sIgA/G - Secretory immunoglobulin A/G
Tm - melting temperature
TSP- Total soluble protein
xxi
VH- Heavy chain variable domain
VL- Light chain variable domain
X-gal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside)
FLW- Fresh leaf weight
mg/g- milligram per gram
µg/g – microgram per gram
xxii
CHAPTER – 1
INTRODUCTION
xxiii
CHAPTER 1
INTRODUCTION
Recombinant monoclonal antibodies are used for various therapeutic and
diagnostic purposes. Genetic engineering of plants for the expression of monoclonal
antibodies has led to the production of “plantibodies” (antibodies produced by plants)
in large scale (Conrad 1998, Kilpatrick 1995, Ma 1996, Fischer 1999, Smith 2000,
Stoger 2002). Genetic manipulation of eukaryotic or prokaryotic DNA by cloning and
transformation, of the known or modified antibody genes in order to produce novel
therapeutic recombinant antibody is of major significance. Plant is an effective
antibody expression system than bacterial and animal expression systems, as they are
cost effective, comparatively safe and easier to scale up antibody production in tons
rather than in kilograms per year (Fischer, 2000). Expression of full-length antibody
and single chain fragment variable (scFv) in plant expression system for cancer
diagnosis and immunotherapy is significant. The antibody-mediated tumor
immunotherapy has become critical in biotherapy and it is used for diagnosis of
cancer, metastasis, fine staging and decisions regarding therapeutic approaches
(Schneebaum, 2000).
xxiv
Hepatocellular carcinoma (HCC) is the third leading cause of cancer death
and the fifth most common malignancy occurring worldwide. Understanding the HCC
biology with recent advances in biotechnology has led to the development of novel
molecular agents targeted for this malignancy. In cancer therapy, antibody fragments
can be applied in the construction of immunotoxins, gene delivery and as anticancer
intrabodies. Antibody fragments directed towards cancer antigens can also be fused
with various toxins such as cytotoxic proteins (Pai, 1998), radionuclides (Liu, 1998) or
drugs (Chari, 1998). The resulting immunotoxins specifically deliver these agents to
cancer antigen presenting cells. These cancer cells are killed when immunotoxins are
internalized. Tumor-specific scFv fused to interleukin-2 has been used for T-cell
mediated eradication of tumors (Thor, 2001). Engineering antibody gene sequence
specific for HCC by cloning and transforming the gene into plant chromosome by
Agrobacterium-mediated transformation is the key tool for HCC-plantibody
production. Producing antibody fragments against HCC in plants to yield higher
amounts of plantibodies is a challenging task. The present research work focuses on
cloning, transforming and expressing a humanized monoclonal antibody (scFv)
specific for HCC in IR-64 rice variety, which will have the capacity to bind
specifically to the receptors of HCC.
xxv
CHAPTER – 2
LITERATURE
REVIEW
xxvi
CHAPTER 2
LITERATURE REVIEW
2.1. PLANTIBODIES
Plant expression system is a cost effective and potential alternative for
antibody production than transgenic animals (Morrow, 2001). Transgenic plant cells
are capable of synthesizing, maturing and assembling the light and heavy polypeptide
chains of the antibody similar to mammalian antibody. A functionally active mouse
antibody was first expressed in tobacco plant wherein the light and heavy chains of the
antibody are correctly folded and assembled in late 1980’s and early 1990’s (Hiatt
xxvii
1989, During 1990). Since the production of the first functional antibody in plants,
many antibodies and antibody fragments have been produced for therapeutic and
diagnostic purposes. Recombinant proteins can be targeted into specific subcellular
compartments or organelles by attaching the signal peptide and KDEL retention signal
which plays a major role in protein accumulation and retention in plants. Glyco-
engineering and humanization of N-glycans can be carried out by mutating the gene
sequence, RNAi interference, retaining the protein in ER, addition of galactose and
sialic acid residues to produce non immunogenic N-glycans. Possibilities of
humanization and glyco-engineering of the plantibodies make plant expression system
as an excellent bioreactor for unlimited production of clinically important antibodies.
The plantibodies are further modified by fusing with enzymes for prodrug therapy,
toxins for cancer, viruses for gene therapy and to target the ligands to specific cellular
targets. It is evident that transgenic plants offers number of advantages for antibody
production over humans, animals, microbes and transfected animals. This review
focuses on plantibody production including glycosylation, glyco-engineering and its
advancements.
2.2. HEPATOCELLULAR CARCINOMA
The incidence of HCC in Asia and Africa is 40 times higher than any other
region in the world due to endemic hepatitis B virus (HBV) infection (Bosch 2004,
Parkin 2001 and Pisani 2002). The major risk factors for HCC development have been
identified as chronic hepatitis B virus (HBV) infection, hepatitis C virus (HCV)
infection, aflatoxin B1 uptake and metabolic disorders, such as hemo-chromatosis and
α1-antitripsin deficiency. In western countries, the incidence of HCC has showed a
sharp increase, mainly because of the high prevalence of hepatitis C virus (HCV)
infection (Fattovich 2004). HCC cases are diagnosed at an early stage and are treated
by surgery (resection or liver transplantation) and loco regional procedures
xxviii
(radiofrequency ablations). Patients diagnosed with early HCC can survive for 5 years.
However after loco regional treatment patients have a poor prognosis due to
underlying liver disease and lack of effective systemic treatment. Application of
monoclonal antibodies against tumor antigens for cancer diagnosis and
immunotherapy is of great importance.
2.3. MONOCLONAL ANTIBODY
Monoclonal antibodies are used as integral components of diagnostic kits
for both screening of asymptomatic individuals, to characterize the stage of tumors and
assessing the tumour response. The property of the monoclonal antibody is their
discrete specificity for a single specific antigenic epitope. A number of monoclonal
antibodies have been produced that are specific for tumor cell antigens. These
antibodies are used in the detection assays to screen the abnormal gene products. The
antibody molecules conjugated with radiolabeled substances provide an alternative
diagnostic tool. Though monoclonal antibodies have extraordinary potential in
diagnoses and treatment, the major disadvantage of using mouse-derived antibody is
the generation of human anti mouse antibody (HAMA) response against mouse-
derived antibody (Osbourn 2003, Krauss 2003). The murine monoclonal antibodies
have short half-life (Kim, 2005) in humans and in cancer patients. Immunological
reactions (Berger, 2002) like allergy (Dass, 2006), serum sickness (Sparrow, 2007)
and renal impairment (Mendez, 1997) have been reported with antibodies of non-
human origin. Genetically engineered antibody such as chimeric antibody or
humanized antibody has shown increased therapeutic efficacy and reduced
immunogenicity during clinical applications.
2.4. FULL LENGTH AND PARTIAL LENGTH ANTIBODY
xxix
The natural immunoglobulin molecule is composed of two heavy and two
light chains. Each chain consists either of one variable and one constant region (light
chain) or one variable and several constant regions (heavy chain). The antigen binding
sites are located in the region of variable protein sequences called the Fv fragment.
Each Fv fragment consists of a heavy chain variable domain (VH) and a light chain
variable domain (VL). These two domains are responsible for specific attachment to
an antigen. Recent advances in recombinant technology have facilitated gene
manipulation, cloning and expression of antibody-encoding genes in various hosts
(Ma, 1998). Recombinant antibodies can be cloned and expressed as intact antibodies,
monovalent antigen binding fragment (Fab), variable-region fragment (Fv) (Hiatt,
1990) and scFv. All of these antibodies and antibody derivatives retain their binding
specificity to the antigenic epitope. One of the most popular antibody forms is scFv
(Ma, 1994). An antibody in scFv format consists of variable regions of heavy and light
chains, which are fused together by a linker sequence. The scFv has been
demonstrated to be particularly useful since it can be encoded by a single gene (which
warrants simple manipulation) and is expressed as a single-function polypeptide chain.
The scFv fragments have the advantage of better penetration into tissues and rapid
clearance than whole immunoglobulins.
2.5. PLANT EXPRESSION SYSTEM -RICE (Oryza sativa)
Selection of a crop is based on a number of factors including overall yield,
in situ protein stability during storage and transport, ease of purification and the cost of
regulatory oversight for the production of plantibodies. While tobacco is favoured
because of its high yield, proteins produced in the tobacco leaf tends to be unstable,
extraction must be carried out immediately to avoid the necessity for post harvest
desiccation or freezing, and processing must also be taken into account for potential
toxic alkaloids. In contrast, cereal seeds provide the ideal environment in which
proteins can accumulate stably and from a regulatory perspective it is generally
xxx
regarded as safe (GRAS) (Sparrow, 2007). Cereal seeds have evolved for protein
storage, allowing the stable accumulation of recombinant proteins in the endosperm.
The endosperm tissue provides the appropriate biochemical environment for protein
accumulation by creating specialized storage compartments such as protein bodies and
storage vacuoles, which are derived from the secretory pathway. Mature cereal seeds
are desiccated, which reduces the exposure of stored proteins to non-enzymatic
hydrolysis and protease degradation. It has been shown that antibodies expressed in
cereal seeds remain stable for several years at room temperature with no detectable
loss of activity (Stoger, 2000).
Rice used for recombinant protein production, shares many advantages with
maize such as the well-established gene transfer technology, its GRAS status, high
grain yield, agricultural and processing infrastructure. Rice is self-pollinating, reduces
gene flow unlike maize. Another difference is that, the endosperm tissue accounts for
more than 90% of the total seed weight (Takaiwa, 2007) and two protein storage
systems are used, namely PB-I and PB-II which facilitate the accumulation of
recombinant proteins (Yamagata, 1986). A comparison of rice with tobacco showed
that rice actually had the higher yield per unit of biomass, although tobacco had the
highest overall yield due to the multiple cropping cycles per year (Stoger, 2002b).
Other crops used as plant expression system are shown in Table 2.5.1. The choice
between rice and maize eventually comes down to local preferences, while rice likely
to dominate in Asia and maize in Africa, Europe and North America. Rice has the
marginal advantage because of its completed genome sequence, which makes it
simpler to identify favourable new regulatory elements and factors affecting yield and
protein stability (Goff, 2002).
xxxi
xxxii
2.6. LITERATURE SURVEY ON PLANTIBODIES
2.6.1. In vivum production of antibodies
xxxiii
An ideal production system must be able to carry out co-and post-
translational modifications, including signal peptide cleavage, pro-peptide processing,
protein folding, disulfide bond formation and glycosylation (Gomord, 2004).
Currently, none of the microbial or mammalian expression system satisfies all these
requirements. The proteins produced in prokaryotes are not properly folded or
processed to provide the desired biological activity. However, for simple therapeutic
proteins such as insulin, interferon or human growth hormones which do not require
folding or extensive post translational processing to be biologically active, microbial
expression system is the choice (Walsh, 2006). Eukaryotic expression systems like
yeast and mammalian cell cultures suffer from many disadvantages in addition to high
operating costs, difficulties in scaling up and potential contamination by viruses and
prions.
Considering the limitations of the prokaryotic and eukaryotic expression
systems and increasing demand for clinical antibodies, plant expression system has
emerged as a suitable alternative expression system with high recombinant protein
production, reproducible quality, efficient and low production cost. It has been well
documented that plant expression system has the capacity to produce high quality
mammalian antibodies which are safe and biologically active (Walsh 2006, Twyman
2003 and Gomord 2004). The production capacity of recombinant antibodies in plant
expression system is unlimited. Recombinant protein production up to 20 kg/ha were
obtained in a plant bioreactor regardless of the plant material (Khoudi 1999, Austin
1994).The usage of transgenic plants could be a solution to meet the rapid increasing
demand for therapeutic antibodies, although expression level of therapeutic proteins
are relatively low (Hiatt 1989, Ma 1995).
2.6.2. Full length and partial antibody produced in plants
xxxiv
Antibodies, produced by the B lymphocytes of the blood plasma cells are
protein molecules that recognize and bind to specific epitopes (termed antigen). The
role of the antibodies is to neutralize and eliminate the antigens. Various classes of
serum antibodies are IgG, IgA, IgM, IgD and IgE. Schematic representation of full-
length and partial antibody is shown in Figure 2.6.2.1
Figure 2.6.2.1: Schematic representation of full length antibody and partial
antibody used for therapeutic antibody production
Full-length antibody bind bivalent antigen and has constant region (Fc-
Fragment constant), which involves in secondary effector mechanisms. CaroRx, the
sIgA/G derived from Guy’s 13 was the first full length plantibody expressed in
tobacco and tested in clinical trials (Ma 1998). Full length antibody production has
been reported in plants by several other groups (Hiatt 1989, Hiatt 1990, During 1990,
xxxv
Ma 1994, Van Engelen 1994). It is also possible to express antibody fragments or
partial antibody rather than the full length antibody and still retain the antigen-binding
specificity (Smith 2000, Chadd 2001 and Fischer 2003). The antigen binding fragment
Fab, F (ab)’ 2 and scFv (single chain variable fragment) are partial antibodies which
can be used for therapeutic antibody production. The Fab and F (ab)’2 fragments
contain only the sequences distal to the hinge region and bind monovalent and bivalent
antigen respectively. The scFv fragments contain variable regions of the heavy and
light chains joined by a flexible peptide chain and bind monovalent antigen. These
fragments can often be effectively used as therapeutic proteins, since they show
increased penetration of target tissues, reduced immunogenicity and are rapidly
removed from the tissues. The scFv antibody specific for Stolbur Phytoplasma has
been expressed in plants to resist against mollicutes, which are wall less bacteria
infecting human, animals and plants (Peeters, 2001). Other derivatives of antibody
include bispecific scFv’s, which contain the antigen recognition elements of two
different antibodies and can bind to two different antigens and scFv-fusions, in which
additional functions are linked to the recombinant proteins (Fischer, 2003). Successful
expression of whole IgGs and smaller antibody fragments, such as Fab, scFv, and
diabody has been demonstrated in plants (Vaquero 2002, Fischer 1999 and Giddings
2000). In tobacco leaves, full length antibodies (IgG) have been expressed at levels of
0.35–1.3% of the total soluble protein (TSP) and scFv at levels between 0.01% - 6.8%.
2.6.3. Antibody gene transformation method
The most commonly used method for the transformation of antibody gene
into plant cell is by Agrobacterium-mediated transformation using Ti-plasmid or by
particle bombardment with DNA-coated gold particles (Hiei 1994). High efficiency
gene transformation has been reported by Agrobacterium tumefaciens (soil borne
bacterium) mediated transformation for the production of fertile and heritable
xxxvi
transgenic plants (Hellens, 2000). Agrobacterium-mediated transformation has several
advantages, which includes higher transformation efficiency, ability to transfer large
piece of DNA with minimal rearrangement, integrate relatively low number of
transgenic copies and stable gene integration into plant cells. Dicotyledonous plants,
like tobacco, are mainly transformed using Agrobacterium tumefaciens. T-DNA
vectors derived from the tumor inducing (Ti) plasmid of A. tumefaciens are used for
gene transformation under the control of viral plant promoter, mostly the constitutive
cauliflower mosaic virus (CaMV35S) promoter. The antibody gene is cloned into T-
DNA region which is flanked by two 25 base pair long imperfect repeats. The T-DNA
transfers the antibody gene from Agrobacterium to the plant nucleus by the action of
virulence (vir) genes in the T-DNA vector (same gene cassette or a separate plasmid)
and subsequently integrates into the plant genome by non-homologous recombination.
Transformed cells are selected by antibiotic resistant markers and complete transgenic
plants are regenerated from the transformed calli (Alamillo, 2006). Rapid transient
antibody production in plants has also been performed using modified plant viral
vectors (Porta 1996, Kikkert 2005) with production levels up to 0.7 % of TSP.
Monocotyledonous plants are mostly transfected by particle gun (Larrick 2001).
Particle bombardment allows the simultaneous introduction of multiple gene
constructs coated on the gold particles into the plant cell by biolistic gun, thereby
expecting the recovery of transgenic lines expressing multimeric antibodies like
secretory immunoglobulin A (sIgA) (Hein, 1991).
Two different approaches have been employed to produce biologically
active whole antibody in plants, a) transformation of the heavy and light-chain genes
separately into plants, followed by cross-pollination by conventional breeding of the
two transgenic parents to yield F1 individuals carrying both the heavy and light chain
genes (Austin 1994, De Neve 1993 and Ma 1994) and b) co- transformation of the
heavy and light chain genes on a single expression cassette (During 1990, Voss 1995
xxxvii
and Yoder 1994). Although co-expressing the heavy and light chain genes on a single
expression cassette may be less involved in cross-pollination strategy, a wide range of
novel antibodies can be produced by combining plant lines carrying different sets of
modifications in either the heavy or light chains (Ma 1994). The co-transformation
system is widely used in Agrobacterium- mediated transformation (Komari, 1996).
This technique is rapid and the problem of segregation in the progenic plants can be
avoided, but the promoter and the terminator genes need to be chosen carefully to
coordinate the expression of the two transgene. An expression level of 1.1% of total
protein was achieved by this technique (Van Engelen, 1994). Super-binary vectors
carrying two separate T-DNAs are used for co-transformation were the plant cell is
transformed with two separate DNAs, one incorporating a gene of interest and the
other containing the selectable marker gene (During, 1990). The efficiency of co-
transformation and the frequency of unlinked integration of the two T-DNAs into
plants were higher through Agrobacterium-mediated transformation method than a
direct gene delivery method.
2.6.4. Protein targeting into plant subcellular compartments
In plant based antibody production, the recombinant protein can be targeted
into specific subcellular compartments, a plant specific strategy to increase the yield
and simplify the purification step by using specific promoters. Recombinant proteins
are targeted to plant cell compartments (endoplasmic reticulum (ER), chloroplast,
vacuole and oil body) for efficient protein expression (Hellwig 2004, Gomord 1999).
High-biomass yield and accumulation of large amount of therapeutic proteins were
obtained when proteins are targeted into various subcellular compartments of the plant
cell.
2.6.4.1. Endoplasmic reticulum
xxxviii
The endoplasmic reticulum (ER) compartment allows the entry of proteins
into the secretory pathway and ensures correct folding and assembly of the newly
synthesized proteins in plants and animals (Van Droogenbroeck, 2008). Large amount
of recombinant protein accumulates in the periplasmic space, implying direct transport
of the proteins from the ER to the periplasmic space between the plasma membrane
and the cell wall. Overproduction of recombinant scFv-Fc disturbs normal ER
retention and protein-sorting mechanisms in the secretory pathway, which is proven by
aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and
the endogenous seed storage protein cruciferin in the periplasmic space (Streatfield,
2006). The recombinant proteins produced so far in plants have been secreted into the
apoplast or intercellular space (Hellwig 2004, Borisjuk 1999). Some recombinant
proteins targeted to the secretory pathway was secreted by the roots into the culture
medium and these proteins accumulated in the medium in higher concentration than in
root tissues (Gaume 2003, Ma 2003) which makes the purification process simple
(Komarnytsky 2006). Recently, active form of antibody has been produced in the roots
of transgenic tobacco (Drake 2003, Sojikul 2003).
2.6.4.2. Protein storage vacuole
Proteins can also be targeted to protein storage vacuole (PSV), an
intracellular organelle of seeds, leaves and roots, where the proteins are stored (Park
2005, Vitale 2005). The PSV of seeds exhibits higher pH value and lower hydrolytic
activity which makes the compartment attractive for recombinant protein accumulation
(Humphrey, 2002). The human lysozyme and human serum albumin have been
expressed in the PSV of rice (Yang 2006, Yang 2003 and Arcalis 2004) and wheat
endosperm (Van Rooijen, 1995), where the protein is biologically active and showed
good stability.
xxxix
2.6.4.3. Oilbodies
Targeting of proteins to oilseeds enables the expression of high level of
proteins and cost effective recovery of therapeutic proteins. The proteins are targeted
to oilbodies as oleosin fusions (Seon, 2002), the major protein at the periphery of the
oilbody membrane, anchored by their hydrophobic domain exposing their N and C
terminal ends to the cytoplasm. The proteins can be purified by simple purification
system, where the proteins are recovered from the oilbodies and other seed
components by liquid- liquid phase separation. This strategy was used to produce
antibodies from different oilseed plants (Staub, 2000).
2.6.4.4. Chloroplast
Expression of proteins in the chloroplast offers several advantages
including very high yield and low proteolytic activity. Plants contain as many as
hundreds of chloroplast in each cell. The transgene coding for the protein can be
introduced into the chloroplast by particle bombardment or homologous recombination
(Daniell, 2002), this stable transformtion allows the amplification of transgene copies
and high accumulation of the recombinant proteins (De Cosa 2001, Faye 2006, and
Daniell 2001). Full length antibodies were produced in transgenic tobacco
chloroplasts, which were able to fold the complex proteins with disulfide bridges
(Mayfield, 2003). Similarly functional antibodies have been produced in the
chloroplast of algae (Cabanes, 1999). Though chloroplast has advantages,
xl
unfortunately chloroplast does not have the capacity to glycosylate proteins which is
essential for therapeutic proteins.
2.6.5. Signal peptide and ER retention signal (KDEL)
Targeting the protein to a specific compartment like ER or vacuoles
requires the addition of signal peptide and ER retention signal (KDEL). The function
of the signal peptide is to lead the protein molecules into the ER lumen for further
processing. In the absence of signal peptide, transgenic plants fail to express light and
heavy chains of the antibody (Austin, 1994). Expression level of recombinant protein
up to 1.3% of total leaf protein has been reported when expressed with signal peptide
at the N- terminus of the gene sequence (Austin, 1994). Recently, it has been reported
that the C-terminal peptide could also affect the expression level of plantibody
production. Expression level of the plantibody increases when the endoplasmic
reticulum retention signal peptide (KDEL) is added to the C-terminus of the scFv (Ma,
1995). Protein retention in the ER is not seen, when the gene sequence is expressed in
the absence of KDEL sequence at C-terminus and signal peptide at N-terminus. The
antibody expression level reached up to 0.01 % of total soluble protein, when the
signal peptide is linked to only N-terminus and 0.2% of total soluble protein, when
KDEL sequence was linked to only C-terminus. Expression level up to 1% of total
soluble protein was obtained when the signal peptide and KDEL sequence were linked
to both N-terminus and C-terminus of the gene sequence (Sriraman, 2004). It was
shown that KDEL -mediated protein retention in the ER could strongly increase the
stability and consequently the protein yield (Verch 1998, Zeitlin 1998, Ramirez 2002,
xli
Wandelt 1992, Tabe 1995, Pueyo 1995, Pagny 2003, Triguero 2005, Takaiwa 2007).
In transgenic alfalfa, accumulation of vicilin, the pea vacuolar storage protein
increases to 100-fold by the addition of ER retention signal (KDEL) to its C-terminus
(Tabe, 1995) and similar results have been obtained in other plant expression systems
(Hood 2007, Gomord 1997). Similar accumulation of storage protein sporamin has
been observed when HDEL, another ER- retention signal was fused to the C-terminus
(Park, 2004).
2.6.6. Protein N-glycosylation in plants
The antibodies produced in plants are proteolytically matured and
glycosylated with high-mannose and biantennary complex type N-glycans (Bakker
2001, Farran 2002).The high mannose-type N-glycans (Man 5-Man9 glycans) have
similar structure as mammalian glycoproteins and are glycosylated at the same Asn
residues, but the complex-type N-glycans remains structurally different from
mammalian glycoproteins. Despite these differences in the structure of N-glycans,
antibodies produced in plants have similar antigen binding specificity as antibodies
produced in mammalian cells (Figure 2.6.6.1).
xlii
Figure 2.6.6.1: N-glycans structure in plants and mammals.
Two classes of N-glycans are synthesized from the same oligosaccharide
precursor attached to the nascent glycoproteins. A. High mannose type N-glycans is
formed from the precursor by glycosidases. The N-glycans structure remains similar in
plants and mammals and contains two N-acetylglucosamine and 5–9 mannosyl
residues. B. The complex type N-glycans are formed from the precursor by the action
of glycosyltransferases. The biantennary complex type N-glycans are structurally
different in plants and mammals. The proximal N-acetylglucosamine core is
substituted by α1, 3 fucose (plants) and α1, 6 fucose (mammals) and the β mannose
core is substituted by a bisecting β1,2 xylose (plants) and a bisecting β1,4 N-
acetylglucosamine (mammals). In addition β1,3 galactose and fucose α1,4 linked to
the terminal N-acetylglucosamine of plant N-glycans and sialic acid in mammals
xliii
instead of β 1,3 galactose.
N-glycosylation of heavy chain determines the binding specificity of the
antibodies and half-life in the bloodstream, which is not affected by the presence of
plant N-glycans instead of mammalian N-glycans (Khoudi 1999, Bouquin 2002 and
Elbers 2001). A mouse immunoglobulin G antibody (MGR48) was expressed in
transgenic tobacco (Nicotiana tabacum cv Samsun NN) to study N-linked
glycosylation. The antibodies isolated from young leaves had relatively high amount
of high-mannose glycans compared with antibodies from older leaves, which contain
more terminal N-acetylglucosamine. The relative amount of N-glycans without
terminal N-acetylglucosamine increased with leaf age (Gomord 2005). Recently
engineering the glycosylated protein to humanize the production of recombinant
protein has been developed to make it clinically useful.
2.6.7. Glyco-engineering and humanization of plantibodies
The addition of α (1, 3)-fucose and β (1, 2)-xylose residues during N-
glycosylation in plants is not seen in mammals, and also they lack terminal galactose
and sialic acid residues, which are found on many native human glycoproteins. The
fucose and xylose residues have been reported to be immunogenic in several mammals
including humans, but curiously not in mice and only after multiple exposures in rats
(Faye 2005, Strasser 2005). Although animal studies indicate the immunogenic impact
of plant-specific glycans, glyco-engineering of the recombinant proteins have been
developed to produce non immunogenic N-glycans by removing the plant-specific
glycans or otherwise ‘humanize’ the glycan profiles of recombinant human
glycoproteins to increase in vivo activity and longevity of therapeutic antibodies
(Figure 2.6.7.1). Various strategies are used to glyco-engineer and humanize
recombinant therapeutic proteins.
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Figure 2.6.7.1: Glycoengineering/ Humanization of plantibodies
Methods to modify plantibodies for clinical therapy
2.6.7.1. Mutating amino acid residues
N-glycosylation can be prevented in the recombinant protein, by mutating
Asn or Ser/Thr residues to inactivate N-glycosylation site. In several plant expression
systems, the plant specific Golgi glycosyltransferases are inhibited by modifying the
enzymatic machinery of the Golgi apparatus to prevent the addition of glyco-epitopes
to recombinant proteins. In Arabidopsis mutants, plant derived glyco-epitopes were
eliminated by Insertional mutation (Downing 2006, Koprivova 2004) and in
xlv
Physcomitrella patens elimination of glyco-epitopes were shown by inactivating the
target gene (Cox 2006). Insertional mutation or gene inactivation to eliminate glyco-
epitopes is done either by knocking out β (1, 2)-xylosylation and α (1, 3)-fucosylation
related genes (Von Schaewen 1993, Frey 2009) and/or by modifying the processing of
glycan structures by adding new glycosyltransferase (Strasser 2008, Misaki 2003). In
Arabidopsis plants, knocking out of the genes coding for β (1, 2)-xylosyltransferase
(XylT) and α (1, 3)-fucosyltranferase (FucT) resulted in the production of N-glycans
with two β-N-acetyglucosamine residues but lacked β (1, 2)-linked xylose and α (1, 3)
linked fucose (Von Schaewen 1993).
2.6.7.2. RNA interference
RNA interference has been used to knock out α 1, 3-fucosyltransferase and
β 1, 2-xylosyltransferase in two plant expression systems, Lemna minor and Medicago
sativa (Sriraman 2004). In Limna minor, plant glycans are humanized by co-
expression of RNAi transcript designed to silence endogenous α (1, 3)-
fucosyltransferase and β (1, 2)-xylosyltransferase activities to produce MDX-060
mAb. The produced mAb has a single major N-glycan species without detectable
plant-specific N-glycans (Sriraman 2004). RNAi technology has also been used for
expression of anti-HIV monoclonal antibody 2G12, to down-regulate the endogenous
XylT and FucT genes in N. benthamiana (Wright 1998.)
2.6.7.3. Protein retention in ER
Plant glycans can be humanized by retaining the recombinant proteins in
ER were the glycans structure remains similar in plants and animals. Further
modification of the glycoprotein in the Golgi apparatus can be avoided by retaining the
recombinant glycoprotein in ER, where plant-specific oligosaccharides are added
xlvi
(Fujiyama 2009). The ER retention signal SEKDEL (Ser-Glu-Lys-Asp-Glu-Leu) or
KDEL (Lys-Asp-Glu-Leu) or HDEL (His-Asp-Glu-Leu) peptides are often used to
retain the protein in endoplasmic reticulum (Tabe 1995, Park 2004, Haq 1995, Ko
2003, Strasser 2004). It has also been shown in tobacco that, by addition of KDEL ER
retention signal at the C-terminal ends of the heavy and light chains contains
exclusively non immunogenic high-mannose type N-glycans (Petruccelli 2006,
Palacpac 1999).
2.6.7.4. Addition of galactose
Plants do not add terminal galactose to the proteins which leads to
immunological reactions (Bakker 2006). It has been demonstrated that the human β1,
4 galactosyltransferase, expressed in plant cells, transfers galactose residues onto the
terminal N-acetylglucosamine residues of plant N-glycans (Misaki 2003, Fujiyama
2001, Jarvis 2003 and Farran 2002). In N. tabacum, human β (1, 4)-
galactosyltranferase (GalT) gene was transferred to add terminal galactose to plant
processed antibody proteins. In another approach, the genes encoding N-terminal
domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human
β (1, 4)-galactosyltransferase I were fused and the chimeric gene expressed in tobacco
plant showed high galactosylation of N-glycan and decline in plant-specific Xyl and
Fuc residues (Kelm 1997).
2.6.7.5. Addition of sialic acid
In the absence of sialic acid residues at the termini of N- glycans, the
circulating proteins get rapidly eliminated from the blood by interacting with
xlvii
galactose-specific receptors on the surface of hepatic cells. Lack of terminal sialic acid
in plant glycoproteins (Fujiyama 2009) makes the proteins short-lived and biologically
inactive (Shah 2003). The production of sialylated N glycans is feasible in plants as
shown in insect cells (Lerouge 1998). Though endogenous sialylated glycol-conjugates
have been found in the plants (Paccalet 2007), it has been found that Neu5Ac, the
major sialic acid present in humans is not synthesized in plants in detectable amounts
(Seveno 2004, Zeleny 2006, Wee 1998). Addition of sialic acid has been successfully
engineered in plants to produce biologically active recombinant proteins. In
Arabidopsis, addition of mammalian α-2, 6-sialtransferase led to the expression of
sialylated N glycans (Misaki 2006). Similarly, addition of human CMP-N-
acetylneuraminic acid synthetase and CMP sialic acid transporter was demonstrated in
tobacco suspension-cultured cells (Saint 2007) and addition of genes encoding
Neu5Ac lyase and Neu5Ac synthase (neuB2) was shown in BY2 cells as well as in
alfalfa (Seveno 2004). Engineering the gene coding for epimerase along with other
components in the same expression system would complete the sialation pathway in
plants (Stein 2001). From this it is clear that plant produced antibodies or proteins can
be glycoengineered or humanized for the production of glycosylated therapeutic
proteins without immunogenic glyco-epitopes.
2.6.8. Successfully produced plantibodies
Several human therapeutic antibodies have been produced in plants.
Currently most of the antibodies and antibody fragments are expressed in tobacco or
corn (Larrick 2001). There are more than 20 companies worldwide that have
developed antibodies in various plant expression systems. The most advanced product
is CaroRX™, produced in tobacco by Planet Biotech (MountainView, CA); a chimeric
secretory antibody drug that has been shown to reduce tooth decay (Yuan 2000).
Epicyte Pharmaceutical (San Diego, CA) is developing several plantibodies to treat
xlviii
inflammatory and infectious diseases. Antibody against herpes simplex virus was
initially produced in soya bean for pre-clinical trials and now in rice expression
systems (Stoger 2002). The other antibodies in Epicyte’s pipeline are being produced
in corn through collaborations with Dow and Dow AgroSciences (Indianapolis, IN).
Meristem Therapeutics (Clermont-Ferrand, France) and Goodwin Biotech (Plantation,
FL) are using tobacco and corn systems to produce therapeutic antibodies. Monsanto
Protein Technologies (St Louis, MO) have also genetically modified corn to produce
monoclonal antibodies.
Chimeric secretory IgG-IgA antibody against surface antigen of
streptococcus mutants, causative agent of tooth decay has been produced in plants and
tested in humans (Torres 1999). Antibody 2G12, one of human immunoglobulin G
(IgG) monoclonal antibody which has the ability to prevent HIV-1 infection in animal
models has been expressed in maize. A humanized antibody, anti-herpes-simplex
virus (HSV) antibody for HSV-2 protein has been produced in soybean, effective in
the prevention of vaginal HSV-2 transmission in a mouse model (Stoger 2000).
Several therapeutic monoclonal antibodies specific for tumor antigens have also been
produced in plants. Antibody against carcinoembryonic antigen (CEA), a tumor-
associated antigen, which is a surface glycoprotein, has been expressed in rice and
wheat (Stoger 2002, McCormick 1999). Antibodies for lymphoma treatment have been
produced in plants (Pujol 2005). Functionally active antibody against anti-hepatitis B
surface antigen has been produced in the seeds of transgenic tobacco plants (Stoger
2000). Other antibodies produced in plants were Co-17 A IgG, scFv for mycotoxin
(Zearalenone), MAK33 IgG1, Fab fragment, MAK33 ScFv, MAK33 Fab and 38C 13
ScFv (Gomord 2004). Several plantibodies have been produced in plant expression
system (Table 2.6.8.1).
xlix
l
li
lii
Secretory immunoglobulin A (sIgA), produced in transgenic tobacco in
1998 (Ma 1995, Ma 1998) is the most extensively studied plantibody and the first
successfully assembled and expressed antibody. At present, six different plant-derived
monoclonal antibodies are being tested and are under clinical trials (Bardor 2003).
CaroRX™ for the prevention of dental carries is in Phase II.
Single-chain Fv antibody fragments produced by viral vectors in tobacco
against non-Hodgkin’s Disease is in Phase I.
IgG (ICAM1) for the prevention of common cold (Phase I)
Antibody against cancer (Phase II)
Rhino RX for the treatment of respiratory syncytial disease (Phase I)
Anti-hepatitis B surface-antigen antibody received regulatory approval in
Cuba for large-scale plant-made antibody production
2.6.9. Current limitations of plantibodies
Though plant expression systems are excellent bioreactors for antibody
production, there are a few limitations like low yield of some therapeutic proteins,
non-mammalian glycosylation, immunogenicity and allergenicity of glycosylated plant
produced antibodies. One of the major limitation to use plant made pharmaceuticals
liii
(PMP) is plant N-glycans harboring specific α (1, 3)-fucose and β (1, 2)-xylose
residues that differs from animal glycoproteins. These non-mammalian glyco-epitopes
have been attributed to elicit immune responses in humans (Jin 2006, Koprivova
2004). Hence controlling the N-glycosylation of plant-made pharmaceuticals remains a
prerequisite for their use in human therapy. To overcome the limitation of plant N-
glycosylation, strategies have been developed to humanize plant derived antibodies by
inhibiting the plant endogenous Golgi glycosyltransferase and/or adding new
glycosyltransferases from mammals (Farran 2002, Bruyns 1996, Streatfield 2006, Haq
1995, Strasser 2004, Von Schaewen 1993, Frey 2009, Strasser 2008) and by retaining
the recombinant glycoproteins in the endoplasmic reticulum (ER), the site where the
plants and animals share the same N-glycan modifications. Another major hurdle is
antibody purification from plants, which can be sorted out easily. A major concern
prevails for the presence of phenols in the plant, which alters the property of the
expressed protein dramatically and irreversibly, which can be removed by
ultrafiltration or diafiltration. Other concerns include secondary metabolites,
endotoxins and mycotoxins, which can be minimized by rapid processing and early
filtration. The removal of these constituents is an important aspect for progressing
towards the clinical trials of plantibodies, which is really not a major obstacle as
several antibodies have been purified to homogeneity for therapeutic use.
Contamination risk by viral pathogens and prions is no longer an issue, but the
regulatory guidelines demand for the screening of herbicide and pesticide residues.
The regulatory authorities are also concerned about the labeling and containment of
the transgenic crops expressing recombinant proteins to prevent the entry of transgene
into the food chain. Strategies for containment include marker selection, self
pollinating crops and male sterility are used.
2.7.0. Future prospects
liv
Plants offer many theoretical advantages over microbial and mammalian
expression systems for the production of antibodies, in terms of cost, safety,
practicality and scalability. Current strategies to improve plant expression systems will
potentially result in increased yield and simplified down-stream processing of
therapeutic proteins. It is interesting to note that several strategies have been
developed to overcome these limitations of plant based antibody production.
Progresses in glyco-engineering, humanization of glycans and reduced heterogeneity
of plant made antibodies are currently making plants as one of the major expression
systems, particularly when large quantities of multimeric recombinant proteins are
required. Although some plant-derived antibody products have successfully completed
early phase clinical trials, several issues including regulatory issues must still be
resolved. A small number of plant derived antibodies have met the technological
challenges, cleared the regulatory hurdles and have approached commercialization.
Owing to the increasing demand of pharmaceutically important therapeutic and
diagnostic proteins, plant expression system will act as an excellent bioreactor for
unlimited antibody production.
lv
CHAPTER – 3
lvi
AIM AND OBJECTIVE
OF THE WORK
CHAPTER 3
AIM AND OBJECTIVE OF THE WORK
AIM
The aim of this research work is to clone, transform and express cDNA of
humanized monoclonal antibody (scFv) against HCC in IR-64 rice variety.
lvii
OBJECTIVES
To clone cDNA of scFv fragment gene against hepatocellular
carcinoma (HCC-scFv) using Vector NTI advance 11.5 software.
To synthesis gene (HCC-scFv) – GENEART.
To transform plasmid (pCAMBIA 1301 and pMA-T) into E. coli
DH5α, by calcium chloride transformation method and to screen the
transformed E. coli DH5α strains.
To isolate the plasmid pCAMBIA 1301 and pMA-T from
transformed E. coli DH5α and confirm by gel electrophoresis.
To clone HCC-scFv from pMA-T plasmid into pCAMBIA 1301
To transform pCAMBIA 1301-HCC-scFv from E.coli DH5α into
Agrobacterium tumefaciens EHA105.
To induce callus in IR64 rice variety for transformation.
To transform pCAMBIA 1301-HCC-scFv from Agrobacterium
tumefaciens EHA 105 into callus by Agrobacterium- mediated
transformation.
To extract total genomic DNA from plant tissues for Molecular
Analysis.
To confirm Putative transformed plants by molecular analysis using
Polymerase Chain Reaction (PCR).
lviii
CHAPTER – 4
lix
MATERIALS
AND
METHODS
CHAPTER 4
MATERIALS AND METHODS
lx
4.1. GENE CLONING USING VECTOR NTI SOFTWARE
4.1.1. OBJECTIVE
To clone cDNA of scFv fragment gene against hepatocellular carcinoma
(HCC-scFv) using Vector NTI advance 11.5 software.
4.1.2. PRINCIPLE
The Vector NTI software contains cloning Tools known as Clone2Seq*
which was designed to recombine two restriction fragments inserted into an
appropriately digested vector. The interface makes it easy to select molecules and
fragments for cloning, to modify 3’ and 5’ ends of the fragment for compatibility and
to create the required recombinant DNA, whether circular or linear. The DNA
fragment can be restricted with various restriction enzymes and the fragments can be
cloned using the software. The cloned fragments are confirmed by electrophoresis
using the software. All cloning functions are present in vector NTI Advance software
including graphical map creation and parent descendant lineage tracking. Any
individual search result can be opened in the Molecule Viewer and sent to Clone2Seq*
for rapid cloning experiment.
4.1.3. MATERIALS
Vector NTI software advance 11.5 version (Invitrogen).
lxi
Gene sequence of anti-HCC scFv antibody (Gene bank) (ref: no: AY686498).
Gene sequence of the plasmid 1301(Gene bank) (ref: no: AF234297).
4.1.4 METHODS
4.1.4.1. Gene sequence of anti-HCC scFv antibody gene: AY686498
Cloning of the anti-HCC scFv antibody gene into plasmid vector
(pCAMBIA 1301) was initially carried out using vector NTI software advance 11.5
version. The gene sequence was downloaded from the Gen bank accession no:
AY686498 (Synthetic construct clone SLH-04-scfv anti-HCC scFv antibody gene).
The sequence of the gene is shown below.
Gene sequence of anti-HCC scFv antibody gene: AY686498
LOCUS AY686498 894 bp DNA linear SYN 26-JUL-2004
DEFINITION Synthetic construct clone SLH-04-scfv anti-HCC scFv antibody gene,
complete cds.
ACCESSION AY686498
VERSION AY686498.1 GI:50428757
KEYWORDS .
SOURCE synthetic construct
ORGANISM synthetic construct
other sequences; artificial sequences.
REFERENCE 1 (bases 1 to 894)
AUTHORS Yu,B., Ni,M. and Shen,G.
TITLE Sceening of special scFv which can ligate to HCC from a large naive
antibody library
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 894)
AUTHORS Yu,B., Ni,M. and Shen,G.
TITLE Direct Submission
JOURNAL Submitted (15-JUL-2004) Tongji Medical College, Department of
lxii
Immunology, Hangkong Road, Wuhan, Hubei 430030, China
FEATURES Location/Qualifiers
source 1..894
/organism="synthetic construct"
/mol_type="genomic DNA"
/db_xref="taxon:32630"
/clone="SLH-04"
CDS 1..894
/codon_start=1
/transl_table=11
/product="anti-HCC scFv antibody"
/protein_id="AAT77091.1"
/db_xref="GI:50428758"
/translation="MKYLLPTAAAGLLLLAAQPAMAQANLRESGPALVKPTQTLTLTC
TFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKYYSTSLKTRLTISKDTSKNQV
VLTMTNMDPVDTAVYYCARHWPTSFDYWGQGTLVTVSSGGGGSGGGGSGGSALEIVMT
QTPLSSPVTLGQPASISFRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKVSNRFSGG
PRQIQWQWGQGQIFTLKNQQGWKLRMSGFITARKLHNFRVGTFGPRDQAGNRRAAAHH
HHHHGAAEQKLISEEDLNGAA"
sig_peptide 1..60
misc_feature 415..462
/note="Region: linker peptide"
misc_feature 823..840
/note="Region: His-tag"
misc_feature 850..891
/note="Region: myc-tag"
ORIGIN
1 atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc
61 atggcccagg ccaacttaag ggagtctggt cctgcgctgg tgaaacccac acagaccctc
121 acactgacct gcaccttctc tgggttttca ctcagcacta gtggaatgtg tgtgagctgg
181 atccgtcagc ccccagggaa ggccctggag tggcttgcac tcattgattg ggatgatgat
241 aaatactaca gcacatctct gaagaccagg ctcaccatct ccaaggacac ctccaaaaac
301 caggtggtcc ttacaatgac caacatggac cctgtggaca cggccgtgta ttactgtgca
361 agacattggc cgacgagttt tgactattgg ggccaaggta ccctggtcac cgtctcgagt
421 ggtggaggcg gttcaggcgg aggtggctct ggcggtagtg cacttgagat tgtgatgacc
481 cagactccac tctcctcgcc tgtcaccctt ggacagccgg cctccatctc cttcaggtct
541 agtcaaagcc tcgtacacag tgatggaaac acctacttga gttggcttca gcagaggcca
601 ggccagcctc caagactcct aatttataag gtttctaacc ggttctctgg gggtcccaga
661 cagattcagt ggcagtgggg gcagggacag attttcacac tgaaaaatca gcaggggtgg
721 aagctgagga tgtcggggtt tattactgca cgcaagctac acaattttcg cgttggtacg
781 ttcgggccaa gggaccaagc tggaaatcga cgtgcggccg cacatcatca tcaccatcac
841 ggggccgcag aacaaaaact catctcagaa gaggatctga atggggccgc ctag
lxiii
4.1.4.2. Modification of Gene sequence of anti-HCC scFv antibody gene:
AY686498
The downloaded gene sequence was further modified to clone the gene into
pCAMBIA 1301 for plant transformation. The modified gene sequence is shown
below.
Gene sequence modification of anti-HCC scFv antibody gene: AY686498-
1 to 951 bp
1 ccatggatgc aggtgctgaa cacgatggtg aacaaacact tcttgtccct ttcggtcctc
61 atcgtcctca tcgtcctctc ctccaacttg acagccggca tggcccaggc caacttaagg
121 gagtctggtc ctgcgctggt gaaacccaca cagaccctca cactgacctg caccttctct
181 gggttttcac tcagcactag tggaatgtgt gtgagctgga tccgtcagcc cccagggaag
241 gccctggagt ggcttgcact cattgattgg gatgatgata aatactacag cacatctctg
301 aagaccaggc tcaccatctc caaggacacc tccaaaaacc aggtggtcct tacaatgacc
361 aacatggacc ctgtggacac ggccgtgtat tactgtgcaa gacattggcc gacgagtttt
421 gactattggg gccaaggtac cctggtcacc gtctcgagtg gtggaggcgg ttcaggcgga
481 ggtggctctg gcggtagtgc acttgagatt gtgatgaccc agactccact ctcctcgcct
541 gtcacccttg gacagccggc ctccatctcc ttcaggtcta gtcaaagcct cgtacacagt
601 gatggaaaca cctacttgag ttggcttcag cagaggccag gccagcctcc aagactccta
661 atttataagg tttctaaccg gttctctggg ggtcccagac agattcagtg gcagtggggg
721 cagggacaga ttttcacact gaaaaatcag caggggtgga agctgaggat gtcggggttt
781 attactgcac gcaagctaca caattttcgc gttggtacgt tcgggccaag ggaccaagct
841 ggaaatcgac gtgcggccgc acatcatcat caccatcacg gggccgcaga acaaaaactc
901 atctcagaag aggatctgaa tggggccgcc aaggatgagc tctagcacgt g
Modifications made to the sequence:
1. Adaptor for NcoI was attached at the 5’end (1st bp- 6
th bp) -ccatgg.
2. Rice α amylase signal peptide was attached at the 5’end (7th
bp-99th
bp).
3. Variable heavy and light chain with linker peptide from 100th
bp-930th
bp.
4. Kdel retention signal peptide was attached from 931th
bp-942th
bp.
5. Adaptor for PmlI was attached at the 3’end (946th
bp -951st bp)- cacgtg.
6. The 24th
code c was replaced by g –cga.
lxiv
The gene sequence (single strand shown in the illustration) from 1to 6 and
23 to 28 base pair (bp) codes for NcoI restriction site and when restricted with NcoI
enzymes, it forms 2 fragments. Therfore the 24th
code c i.e. cca coding for theronine
was replaced by cga which also codes for the same amino acid theronine. This
sequence modification will help to clone the gene into pCAMBIA 1301 at NcoI and
PmlI, were the entire cDNA strand will remain unrestricted between 7th
bp to 945th
bp.
The rice α amylase signal peptide was added at 5’end (ATG CAG GTG
CTG AAC ACC ATG GTG AAC AAA CAC TTC TTG TCC CTT TCG GTC
CTC ATC GTC CTC ATC GTC CTC TCC TCC AAC TTG ACA GCC GGC
ATG) to target the protein into the Endoplasmic reticulam (ER) and KDEL retention
signal peptide at 3’ end (AAGGATGAGCTC) to retaining the protein in
Endoplasmic reticulum(ER).
The modified gene sequence was cloned into pCAMBIA 1301 T-DNA
plasmid vector sequence restricted with NcoI and PmlI restriction enzymes and
ligated. The cloned construct was further restricted with the same NcoI and PmlI
restriction enzymes and confirmed by running electrophoresis using the software. All
the steps were carried out in the software, prior to gene synthesis.
lxv
4.2 SYNTHESIS OF HCC-scFv GENE-GENEART
4.2.1. OBJECTIVE
To synthesis modified AY686498: gene (HCC-scFv) sequence –
GENEART.
4.2.2. METHOD
The modified gene squence confirmed by the analysis of Vector NTI
software for cloning was sent for gene synthesis to GENEART, Germany. The
stability of the gene strand was analyzed with Gene Optimizer for G+C content. When
the G+C content was confirmed to be above 50%, the gene synthesis was initiated. The
synthesized gene was confirmed by gene sequencing and cloned into a standard
plasmid vector pMAT-T (3329 bp) containing ampicillin resistance marker with colE1
replicon, using Sfil and Sfil cloning sites. Plasmid containing the synthesized gene of
interest of 5 µg was delivered in lyophilized form.
lxvi
4.3. TRANSFORMATION OF PLASMID pCAMBIA 1301 AND pMA-T INTO
E. coli DH5α AND SCREENING OF TRANSFORMED E. coli DH5α STRAIN
4.3.1. OBJECTIVE
To transform plasmid (pCAMBIA 1301 and pMA-T) into E. coli DH5α, by
calcium chloride transformation method and to screen the transformed E. coli DH5α
strains.
4.3.2. PRINCIPLE
Certain species of bacteria naturally take up the DNA at certain stage of
growth. Transformation is the uptake of naked DNA by an organism and competence
is the state of being able to take up the DNA. Certain bacterial species have natural
transformation system where bacterial cells acquire competence for DNA uptake.
Some species are not naturally competent at any stage of growth. In such cells,
competence can be artificially induced by treating them with calcium chloride (Dagert
1979, Hiroaki 1990).
The exact mechanism of plasmid DNA uptake by competent E.coli cells is
unknown. Unlike salts and small organic molecules such as glucose, DNA molecules
lxvii
are too large to diffuse or transport through the cell membrane. Some bacteria possess
membrane proteins that recognize the DNA and facilitate the absorption of short DNA
sequences derived from the relative species. One hypothesis states that DNA molecule
pass through any of several hundred channels formed at adhesion zones, where the
outer and inner cell membranes are fused to pores in the bacterial cell wall.
The fact that the adhesion zones are only present in growing cells, in
consistent with the observation that the cells in logarithmic phase can render most
competence for plasmid uptake. However, acidic phosphates of the DNA helix are
negatively charged, as well as the phospholipid composition of the cell membrane and
the membrane pores are negatively charged. Thus electrostatic repletion between
anions may effectively block the movement of the DNA through the adhesion zones.
Analysis of this ionic interaction produces a possible hypothesis for DNA
uptake in bacteria. Treatment of the cells at 0°C crystallizes the fluid cell membrane,
stabilizing the distribution of charged phosphates. The cations in the transformation
solution (Ca++
,Mn++
,K+,Co
+++) forms complex with exposed phosphate groups,
shielding the negative charges. With this ionic shield in place, a plasmid molecule can
then move through the adhesion zone. Heat shock complements this chemical process
perhaps by creating a thermal imbalance on either side of the E.coli membrane that
physically helps to pump the DNA molecule through the adhesion zone.
The transformed plasmids are selected based on the presence of antibiotic
marker genes present on the plasmid. The transformed pCAMBIA 1301 was screened
based on kanamycin antibiotic and the pMA-T was screened based on ampicillin
antibiotic. The plasmid pCAMBIA 1301 was also screened for the presence of lacZ
gene.
4.3.3. MATERIALS
lxviii
pCAMBIA 1301
pCAMBIA Legacy Vector Kit obtained from CAMBIA, Australia. For cloning,
pCAMBIA 1301was used as plasmid vector- Appendix 2.
HCC-scFv-pMA-T
The synthesized gene was inserted into pMA-T vector and supplied by GENEART in
lyophilized form (5µg of plasmid).
Recipient strain
E. coli DH5α obtained from Madurai Kamaraj University (MKU).
2X YT medium (200 ml) pH 7.0
Tryptone 3.2g
Yeast Extract 2.0g
Nacl 1.0g
Mgcl2.6H2o 0.8132g
LB medium (1000 ml) pH 7.2
Tryptone 10.0g
Yeast Extract 5.0g
Nacl 10.0g
Agar (1.5%) 15.0g
100mM Cacl2 (400ml)
lxix
Weigh 5.88 g of Cacl2 (1M=147.01g/mol-Merck: 10238005001730) and dissolve in
350 ml of autoclaved millipore water. Make up the volume to 400ml. Autoclave and
store at 4°C.
X-gal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside)- Sigma B4252
Stock: (20mg/ml): 20mg of X-gal was dissolved in DMF (dimethyl formamide). The
tube was wrapped in aluminum foil and stored at -20°C.
Working stock: X-gal was used in plates at a concentration of 40µg/ml: 2µl/ml of LB
media.
IPTG (Iso propyl thiogalactoside or isopropyl beta-D-thiogalactopyranoside)-
Sigma-11502
Stock: 0.1 M IPTG :( 1M=238.3g/mol): 0.2383 g of IPTG was dissolved in 10 ml of
autoclaved Millipore water and stored at -20◦C.
Working stock: IPTG was used in the concentration of (0.1mM): 1µl/ml of LB media.
Kanamycin
Stock: (100mg/ml)-Kanamycin of 100mg was dissolved in 1 ml of sterile autoclaved
Millipore water.
Working stock: (50µg/ml)
Ampicillin
Stock: (100mg/ml)-Ampicillin of 100mg was dissolved in 1ml of sterile autoclaved
Millipore water.
Working stock: (100 µg/ml)
4.3.4. METHOD
lxx
4.3.4.1. Plasmid preparation of pCAMBIA 1301vector
The paper containing the plasmid was cut and placed in 0.5 ml centrifuge
tube pierced with a needle at the base, and placed over a 1.5 ml eppendorf tube. TE
buffer of 50µl was added and incubated at room temperature for 10 minutes. The
content was spinned down into 1.5 ml eppendorf tube and stored at -20◦C until
transformation. The concentration of plasmid was checked using Biophotometer
(Germany).
4.3.4.2. HCC-scFv-pMA-T vector
Lyophilized plasmid of 5µg was dissolved in 50 µl of sterile syringe water
and stored at -20◦C until transformation.
4.3.4.3. Competent cell preparation
A single colony of E. coli (DH5α) was inoculated into 2 ml of LB culture
broth and incubated (Scigenics, India) at 37°C for 10 hours to 12 hours overnight.
From the overnight culture, 500µl was inoculated into 50 ml of 2X YT medium and
incubated at 37°C in a rotary shaker until it reached an O.D of 0.5 to 0.6 at 600nm
(Biophotometer, Germany). To a sterile 50 ml polypropylene tube, 30 ml of the culture
was transferred and incubated in ice for 30 minutes. The culture was centrifuged at
5000 rpm at 4°C for 10 minutes. The supernatant was discarded and the pellet was
resuspended in a small volume of medium left behind. Finally the pellet was gently
resuspended in 30 ml of ice cold 100mM CaCl2 and incubated in ice for 30 minutes.
The resuspended cells were centrifuged at 5000 rpm for 10 minutes at 4°C. The
lxxi
supernatant was discarded and the pellet was resuspended gently in 3ml (1/10th
volume
of CaCl2) of ice cold 100mM CaCl2.The competent cells were stored in ice for at least
30 minutes before use.
4.3.4.4. Transformation of competent cells (E. coli)
Competent cells of 200 µl was aliquated into 2ml sterile eppendorf tubes
and incubated in ice (cut tips were used to aliquot the cells). Plasmid pCAMBIA 1301
1301 and pMA-T-HCC-scFv of 200ng was dispensed into the above eppendorf tubes
containing competent cells separately and incubated in the ice for 30 minutes. Heat
shock was given for 2 minutes at 42°C in water bath and then transferred and
incubated in ice for 10 minutes. The cells were transferred to 0.8 ml of LB media and
incubated at 37°C in a rotary shaker for 1 hour at 220rpm.The LB mixture was then
centrifuged at 10,000 rpm for 5 minutes and the supernatant was discarded.
From the pellet, 100µl of the cells were plated onto LB agar plate
containing antibiotics kanamycin (50µg/ml) for pCAMBIA 1301 and 200 µl of the
cells were plated onto LB agar plate containing Ampcillin (100 µg/ml) for pMA-T-
HCC-scFv. The plates were incubated overnight at 37°C. In case of pCAMBIA 1301,
the transformed colonies were further screened for the presence of blue colonies by
streaking the transformed cells onto LB medium containing X-gal (40µg/ml) and IPTG
(0.1mM).
lxxii
4.4. ISOLATION OF PLASMID pCAMBIA 1301 AND pMA-T FROM
TRANSFORMED E. coli DH5α AND CONFIRMATION BY GEL
ELECTROPHORESIS
4.4.1. OBJECTIVE
To isolate plasmid pCAMBIA 1301 and pMA-T from transformed E.
coli DH5α and to confirm by gel electrophoresis.
4.4.2. PRINCIPLE
A rapid method for making a small preparation of purified plasmid DNA
from bacterial culture as low as 1ml is called “miniprep”. The alkaline lysis method is
a modified method of Birnbroim and doly (1979) and Ish.horowwicz and burkes
lxxiii
(1981). In this method the cells are lysed and the DNA is denatured by high pH of
solution II. The Solution III returns the lysis mixture to the neutral pH. The plasmid
DNA which remains partially intact, renatures rapidly than the chromosomal DNA
fragments, which gets pelleted along with the denatured proteins. The RNA which
remains along with the plasmid in the supernatant is removed by RNase treatment. The
Plasmid can then be concentrated by the addition of sodium acetate salt and 95 %
ethanol. Based on the salting out principle the sodium acetate salt removes the water
molecule, which causes the double stranded plasmid DNA to aggregate and
precipitate.
4.4.2.1. Principle behind the reagents used
Solution I: It acts a buffer in which glucose increases the osmotic pressure of the cell,
EDTA removes the magnesium ions that are essential for preserving the overall
structure of the cell membrane and also inhibits the cellular enzymes that could
degrade DNA and Tris helps in maintaining the cytoplasmic pH of the cell.
Solution II: The alkanine mixture (SDS/NaOH) lysis the bacterial cells. The detergent
SDS dissolves the lipd components of the cell membrane, as well as the cellular
proteins. The NaOH denatures the chromosomal and plasmid DNA into single strands.
The intact circles of plasmid DNA remain intertwined.
Solution III: The acetic acid returns the pH to netural, allowing DNA strands to
renature. The large disrupted chromosomal strands cannot rehybridize perfectly, but
instead collapse into a partially hybridized tangle. At the same time, potassium acetate
precipitates the SDS from the cell suspension along with the protein and the lipids.
The renaturing chromosomal DNA is trapped in the SDS/ Lipid / Protein precipitate.
Only smaller plasmid DNA and RNA molecules escape the precipitate and remain in
the solution.
lxxiv
Isopropanol: The isopropyl alcohol rapidly precipitates the nucleic acid but slowly
precipitates the protein. The organic solvent content increases as the availability of the
water decreases. Thus the solubility of the DNA comes down and plasmid DNA gets
pelleted.
Tris / EDTA: Tris buffers the DNA solution, EDTA protects the DNA from
degradation by DNase by binding divalent cations that are necessary co factors for
DNase activity, buffering DNA is important as low pH(<6) leads to the loss of purines
(adenine and guanine) called depurination.
4.4.3. MATERIALS
Solution I: 100ml (pH8.0)
M.wt for 100ml
Tris (25mM) - 121.1 0.303g
EDTA (10mM) - 372.0 0.372g
Glucose (50mM) -180.16 0.901g
lxxv
The salts were weighed and dissolved in 80 ml of millipore water and the pH was
adjusted to 8.0 using 1 N HCl. The volume was made up to 100 ml. Autoclaved and
stored at room temperature.
Solution II: (Prepared fresh every time)
NaOH (0.2 M)
SDS (1.0 %)
NaOH (0.4 N) was prepared and stored in a plastic reagent bottle. SDS (2%) was
prepared and autoclaved. The solutions were mixed in the ratio of 1:1 before use.
(NaOH- was not autoclaved)
Solution III: 100ml (pH 5.5)
3 M potassium acetate
Glacial acetic acid
For 100 ml, 29.4 gm of potassium acetate was weighed and dissolved in 25 ml to 30
ml of autoclaved millipore water. The pH was adjusted with glacial acetic acid and the
volume was made up to 100ml. Autoclaved and stored at 4°C.
RNase-(Cat- RD0187)
Stock: 10mg/ml
Working stock concentration: 1 µl
RNase (Fermentas) was treated initially by heating to 100 °C for 15 minutes in boiling
water bath (to denature DNase) and allowed to cool to room temperature and stored at
-20°C.
TE(0.1X) pH 8.0
Tris HCl (1mM)
EDTA (0.1mM)
lxxvi
For 100ml, 100µl from 1 M stock (pH 8.0) and 20 µl from 0.5 M stock (pH 8.0) was
added and made up to 98.8 ml with sterile autoclaved milli Q water.
Chloroform: Isoamyl alcohol
Chloroform: Isoamyl alcohol was prepared in the ratio of 24: 1.
3M sodium acetate:100ml (pH 5.2)
24.61 g of sodium acetate was weighed and dissolved in 80 ml of autoclaved millipore
water. The pH was adjusted with glacial acetic acid. The volume was made up to 100
ml. Autoclaved and stored at 4°C.
70% Ethanol
To sterile 30ml of autoclaved Millipore water 70 ml of absolute ethanol was added and
mixed.
10X TBE: 1000ml (pH8.2)
Tris : 107.78g
EDTA: 8.41g
Boric acid :55.00g
The above chemicals were dissolved in 600ml of autoclaved water and made upto one
liter and autoclaved.
Ethidium Bromide
Stock: 10mg/ml
Working stock: 1µg/ml.
Agarose gel preparation (0.7%)
lxxvii
In a 50 ml 1X TBE buffer 0.35g of agarose was added, melted in microoven for 3
minutes, cooled to about 44°C to 50°C and poured into a gel platform with the comb in
position.
Running gel
After the solidification of the gel (approx.45 min), the gel was placed in a gel tank
with 1X TBE buffer. The buffer was filled to the surface of the gel. The samples were
loaded in each well and the electrophoresis was carried out at 60V till the blue dye
runs to the end.
Staining the gel
Staining solution was prepared by adding 10 µl of 10mg/ml stock of Ethidium bromide
in 100ml of sterile autoclaved millipore water.
Gel loading dye: 10X stock (10ml)
Bromophenol blue – 0.25%
Ficoll - 25%
Bromophenol blue of 25mg was weighed and dissolved in 7 ml of sterile autoclaved
Millipore water, in screw cap centrifuge tube. Ficoll of 2.5 g was added and dissolved
completely (kept in shaker, overnight). The volume was measured and made up to
10ml using autoclaved Millipore water and stored at 4°C.
4.4.4. METHOD
lxxviii
4.4.4.1. Plasmid isolation by alkaline lysis method
The isolation of plasmid was carried out following the steps given below.
1. Transformed E. coli DH5α (pCAMBIA 1301) and E. coli DH5α (pMA-T) was
inoculated into 10 ml of LB broth containing Kanamycin and Ampicillin antibiotic
for 4-5 hours at 37 °C in a rotary shaker till late log phase.
2. In 1.5 ml eppendorf tubes, 1.5 ml of the culture was taken and centrifuged at 12,000
rpm for 5 minutes and the supernatant was discarded. Another 1.5 ml culture was
centrifuged in the same eppendorf tube and the supernatant was discarded.
3. The pellets were resuspended in 100 µl of Solution I (the cells were suspended well
using vortex mixer).
4. Immediately 200 µl of freshly prepared Solution II was added. The contents were
mixed by inverting the tubes 4 to 6 times and kept in ice for 15 minutes.
5. To each tube 150 µl of ice cold Solution III was added and gently mixed by
inverting the tubes upside down and kept in ice for 15 minutes.
6. The tubes were centrifuged in a microfuge for 15 minutes at 12,000 rpm.The
supernatant was collected and 300 µl of neutral phenol was added. The content was
mixed and microfuged for 5 minutes.
7. The aqueous phase was carefully transferred into fresh eppendorf tube avoiding
interphase.
lxxix
8. Ice cold isopropanol of 300 µl was added, mixed and left in the room temperature
for 2 minutes.
9. The content was centrifuged at 12,000 rpm for 10 minutes. The supernatant was
discarded and drained over tissue paper for 5 minutes. To the pellet 100 µl TES was
added and the pellets were dissolved.
10. Neutral phenol: chloroform mix of 150 µl was added and spinned for 5minutes.
11. Aqueous phase was collected and ether saturated with water was added (200 µl to
250 µl) mixed and spinned for 5 minutes.
12. The top ether phase was removed and ether extraction was repeated.
13. The tubes were kept open in the water bath for 10 minutes at 50°C to remove
ether.
14. The ether extracted aqueous phase was measured and 1/10th
volume of 3 M sodium
acetate (pH 5.2) was added.
15. To the above aqueous phase 2.5 volumes of 95% ethanol (ice cold) was added and
mixed by inverting the tubes.
16. The tubes were kept in -20°C overnight.
17. The tubes were microfuged at 12,000 rpm for 10min at 4°C.
lxxx
18. Ethanol was decanted and 500 µl of 70% ethanol was added to the pellet. Gently
the tubes were mixed by inverting and spinned at 12,000 rpm for 5 minutes at 4°C.
19. The pellets were dried for 10 minutes.
20. The dried pellets were dissolved in 20 µl of 0.1 X TE (pH 8.0).
21. The presence of the plasmid DNA was checked by loading 5 µl on agarose
minigel.
Note: some samples were treated with Heat treated RNase of 1 µl (10mg/ml) was
added and incubated in a water bath at 37°C for 5 minutes after step9.
4.4.4.2. Gel electrophoresis
Gel electrophoresis was carried out following the steps given below.
1. Gel was casted using agarose (8.0%). Agarose was weighed and dissolved in 1X
TBE buffer by heating in a microwave oven until the agarose melted. After the
solution was cooled to about 60°C, it was poured into a casting tray containing a comb
and allowed to solidify at room temperature for 45 minutes.
2. The comb was carefully removed after the gel was solidified without ripping the
bottom of the gel. The casting tray along with the gel was placed horizontally in the
electrophoresis tank containing 1X TBE buffer.
3. The plasmid samples (5 µl) and gel loading dye (3 µl) was added to the wells and
electrophoresis was carried out at 60V till the blue dye runs 75% of the gel.
lxxxi
3. The gel was placed in a staining solution containing ethidium bromide for 30
minutes and viewed in UV transilluminator.
4. The image of gel was captured using gel documentation and the gel was analysed
in Biorad and Biocaputre software.
lxxxii
4.5. CLONING OF HCC-scFv FROM pMA-T PLASMID INTO pCAMBIA 1301
4.5. PRINCIPLE
Cloning the gene of interest into plasmid vector requires restriction
enzymes and T4 DNA ligase. Cloning of HCC-scFv from pMA-T into pCAMBIA
1301 plasmid is shown in the Figure 4.5.1.
lxxxiii
Figure 4.5.1: Illustration of cloning HCC-scFv gene into pCAMBIA 1301.
4.5.1. RESTRICTION DIGESTION OF pCAMBIA 1301 AND pMA-T FOR
CLONING
4.5.1.1. OBJECTIVE
To restrict digest pMA-T (HCC-scFv) and pCAMBIA 1301 (Ti-binary-T-
DNA vector) using restriction enzymes.
4.5.1.2. PRINCIPLE
RESTRICTION ENZYMES
Restriction enzymes are endonucleases that cleave the deoxy ribose sugar-
and phosphate backbone of the DNA. This leaves a phosphate group on the 5' ends and
a hydroxyl on the 3' ends of both strands. Restriction enzymes recognise 6 to 8 bp on
double stranded DNA and usually recognise palindrome sequence. Type II restriction
enzymes recognise specific DNA sequence and cleave the DNA at the specific
recognition site or near to the recognition site. Fast digest enzymes are an advance line
of restriction enzymes for rapid DNA digestion. The fast digest enzymes are able to
digest the DNA in 5-15 minutes. This enables any combination of restriction enzymes
to work simultaneously in one reaction tube and eliminates the need for sequential
digestions. These enzymes can be used for digestion of plasmid, genomic, PCR and
viral DNA and does not show star activity even in prolonged incubations. The
enzymes used in this experiment are PmlI and NcoI.
lxxxiv
PmlI recognises the sequence given below
NcoI recognises the sequences given below
PmlI causes blunt end restriction and NcoI causes sticky end restriction. One unit of
enzyme is usually defined as the ability to digest 1 µg of lambda DNA with 1µl of the
enzyme in 5 minutes at 37°C in 20 µl of reaction mixture.
4.5.1.1. MATERIALS
Restriction enzyme
1. Fast Digest® NcoI (FD0574)-Fermentas
2. Fast Digest® PmlI (Eco721)(FD0364)- Fermentas
Eppendorf tube
Biophotometer
Water bath
TAE buffer (50X)-pH: 8.0
Stock: -100ml (autoclaved Millipore water)
Tris Base (MW: 121.1) - 24.20g
EDTA (MW: 372.2) -1.86g
lxxxv
Glacial acetic acid -5.71ml
Working stock (500ml)
To 490ml of autoclaved Millipore water, 10 ml of TAE buffer (50X) was added and
the buffer was used for 0.8% agarose gel preparation and as buffer for electrophoresis.
4.5.1.2. METHOD
1. The following reaction components were combined at room temperature in the
order indicated in Table 4.5.1.1 and Table 4.5.1.2. The reaction volume was
increased based on the amount of plasmid DNA used for restriction digestion.
Water,nuclease-free(R0581) 14µl 24µl
10 X Fast Digest® 2 µl 4 µl
Plasmid pCAMBIA1301 2 µl(up to1µg) 4 µl (4 µg/ µl)
Restriction enzyme Fast Digest® NcoI 1 µl 4 µl
Fast Digest® PmlI 1 µl 4 µl
Total volume 20 µl 40 µl
Table 4.5.1.1: Restriction Digestion set up: pCAMBIA 1301: Vector DNA
Water,nuclease-free(R0581) 14µl 24µl
10 X Fast Digest® 2 µl 4 µl
Plasmid pMA-T (HCC-
scFv)
2 µl(up to1µg) 4 µl (4 µg/ µl)
lxxxvi
Restriction enzyme Fast Digest® NcoI 1 µl 4 µl
Fast Digest® PmlI 1 µl 4 µl
Total volume 20 µl 40 µl
Table 4.5.1.2: Restriction Digestion set up: pMA-T (HCC-scFv): Insert DNA
2. The mixture was gently mixed and spinned down in microfuge.
3. The tubes were incubated at 37°C (both enzymes restrict at the same time) in a
thermostat water bath for 10 minutes or15 minutes (when the volume exceeds 20 µl).
4. Digested sample of 5 µl was loaded onto 0.8% agarose gel prepared in TAE
with 3 µl of gel loading dye and electrophoresis was carried out at 60V till the
blue dye runs 75% of the gel.
5. The gel was placed in a staining solution containing Ethidium bromide for 30
minutes and viewed in UV transilluminator.
6. The image of gel was captured using gel documentation and the gel was
analyzed in Biorad and Biocaputre software.
lxxxvii
4.5.2. LIGATION OF HCC-scFv AND pCAMBIA 1301
4.5.2.1. OBJECTIVE
To ligate restrict digested fragments using T4 DNA ligase enzyme.
4.5.2.2. PRINCIPLE
T4 DNA ligase enzyme catalyzes the formation of a phosphodiester bond
between 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA (Weiss and
Richardson (1967), Lehnman 1974). This enzyme will join blunt end and cohesive end
termini and also repairs single stranded nicks in duplex DNA, RNA or DNA/RNA
hybrids. T4 DNA ligase requires ATP as the cofactor for ligation reaction, whereas E.
coli DNA ligase requires NAD as the cofactor. Ligation reaction was carried out at the
room temperature (20°C to 25°C). The Rapid DNA ligation kit enables sticky-end or
blunt-end DNA ligation in only 5 minutes at room temperature. The kit includes T4
lxxxviii
DNA Ligase and a specially formulated 5X Rapid Ligation Buffer optimized for fast
and efficient DNA ligation. The ligation reaction mixture can be used directly for
bacterial transformation using calcium chloride transformation method or bacterial
transformation kit. The transformed ligated clone was confirmed on 0.8% agarose gel
electrophoresis (Adkins 1996).
4.5.2.3. MATERIALS
4.5.2.3.1. GEL ELUTION OF THE PARTICULAR BANDS RESTRICTED
WITH RESTRICTION ENZYMES
Gel elution Kit (Real Genomics, Hi Yield TM
Gel/PCR DNA MINI KIT-
YDF100/YDF300)
100 mini preps/kit
DF Buffer: 80ml
W1 Buffer: 45ml
Wash Buffer (concentrated):25ml*
Elution Buffer: 6ml
DF Column: 100pcs
2ml Collection Tube: 100pcs
*Initially 100ml of ethanol (96-100%) was added to 25 ml of wash buffer prior to use
Sterile Lancet Blade
Plastic cover
Scale
2ml eppendorf tubes
DNase-free pipette tips
4.5.2.3.2. LIGATION OF THE VECTOR (pCAMBIA 1301) AND INSERT
(HCC-scFv)
lxxxix
Rapid DNA Ligation Kit (K1422- Fermantas)
Linearized vector DNA- (pCAMBIA 1301-9.802 kb)
Insert DNA - (at 3:1 molar excess over vector)-(HCC-scFv-0.946 kb)
5X Rapid Ligation Buffer
T4 DNA Ligase (5U/ µl)
Water, nuclease-free
Autoclaved microfuge tubes, microtips etc.
4.5.2.3.3. TRANSFORMATION OF THE LIGATED SAMPLES
2X YT medium (200 ml) pH 7.0
Tryptone 3.2g
Yeast Extract 2.0g
Nacl 1.0g
Mgcl2.6H2o 0.8132g
LB medium (1000 ml) pH 7.2
Tryptone 10.0g
Yeast Extract 5.0g
Nacl 10.0g
Agar (1.5%) 15.0g
100mM Cacl2 (400ml)
xc
Weigh 5.88 g of Cacl2 (1M=147.01g/mol-Merck: 10238005001730) and dissolve in
350 ml of autoclaved millipore water. Make up the volume to 400ml. Autoclave and
store at 4°C.
X-gal (5-Bromo-4-chloro-3-Indolyl-β-D-galactoside)-Sigma B4252
Stock: (20mg/ml): 20mg of X-gal was dissolved in DMF (dimethyl formamide).The
tube was wrapped in aluminum foil and stored at -20°C.
Working stock: X-gal was used in plates at a concentration of 40µg/ml: 2µl/ml of LB
media.
IPTG (Iso propyl thiogalactoside or isopropyl beta-D-thiogalactopyranoside-
Sigma-11502
Stock: 0.1 M IPTG :( 1M=238.3g/mol): 0.2383 g of IPTG was dissolved in 10 ml of
autoclaved Millipore water and stored at -20◦C.
Working stock: IPTG was used in the concentration of (0.1mM): 1µl/ml of LB media.
Kanamycin
Stock: (100mg/ml)
Kanamycin of 100mg was dissolved in 1 ml of sterile autoclaved Millipore water.
Working stock: (50µg/ml)
4.5.2.4. METHOD
4.5.2.4.1. GEL ELUTION OF THE PARTICULAR BANDS RESTRICTED
WITH RESTRICTION ENZYMES
Gel Dissociation
xci
1. The agarose gel containing the samples restricted with enzymes was placed on a
plastic paper over the UV illuminator. The bands were visualized and particular
band of interest was marked.
2. The band of particular size of approximately 10Kb (pCAMBIA1301) from lane
2-5 and approximately 1Kb (HCC-scFv-pMA-T) from lane6-9 was excised
from the gel using sterile lancet blade.
3. Extra agarose gel was removed to minimize the size of the gel. The gel was
made using TAE buffer as TBE buffer might affect the downstream experiment.
4. The gel slice of 300mg was transferred into 1.5 ml microcentrifuge tube.
5. DF Buffer of 500µl was added to the samples and mixed by vortexing.
6. The tubes were incubated at 55 to 60°C for 10-15 minutes until the gel slice
have been completely dissolved. The tubes were inverted every 2-3 minutes.
7. The dissolved samples were cooled to room temperature.
DNA Binding
8. DF column was placed in a 2 ml collection tube.
9. The sample mixture of 800µl from step 7 was added into DF Column.
10. The column was centrifuged at full speed (approximately 13, 000 rpm) for 30
seconds.
11. The flow through was discarded and the DF Column was placed back into the 2
ml collection tube. When the samples were more than 800µl, this step was
repeated.
Wash
12. W1 Buffer of 400 µl was added into the DF Column.
13. The columns were centrifuged at full speed (approximately13, 000 rpm) for 30
seconds.
xcii
14. The flow through was discarded and the DF column was placed back into the 2
ml collection tube.
15. Wash buffer of 600 µl was added into the DF column and allowed to stand for 1
minute.
16. The column was centrifuged at full speed (approx.13, 000 rpm) for 30 seconds.
17. The flow through was discarded and the DF column was placed back in 2ml
collection tube.
18. The columns were centrifuged again for 3 minutes at full speed (approximately
13000rpm) to dry the column matrix.
DNA Elution
19. The dried DF column was transferred into a new 1.5ml microcentrifuge tube.
20. Elution buffer or TE of 20 µl to 50 µl was added into the center of the column
matrix.
21. The elution buffer was allowed to stand for 2 minutes until the buffer is
absorbed by the matrix.
22. The column was centrifuged at full speed for 2 minutes to elute the purified
DNA
23. The eluted DNA of 2 µl was diluted with 48 µl of sterile water and the quantity
was measured using biophotometer.
4.5.2.4.2. LIGATION OF pCAMBIA 1301 FRAGMENT AND Hcc-scFv
FRAGMENT OF pMA-T
1. The 5 X Rapid ligation buffer was thoroughly mixed prior to use.
2. The ligation reaction was setup as below (Table 4.5.2.1) in a microfuge tube.
xciii
S. No Reaction mixture volume
1 Linearized vector DNA
(pCAMBIA 1301-9.802 kb)
2 µl (100ng)
2 Insert DNA(at 3:1 molar excess over vector)
(HCC-scFv-0.946 kb)
1 µl (28.95ng )
(approx. 29ng)
3 5X Rapid Ligation Buffer 4 µl
4 T4 DNA Ligase(5U/ µl) 1 µl
5 Water, nuclease-free 12 µl
Total volume 20 µl
Table 4.5.2.1: Setup of Ligation reaction
3. The vector and insert DNA ratio was calculated using the Equation (4.5.2.1).
Vector: Plasmid (pCAMBIA 1301) - concentration of plasmid used was 100ng and it
was about 9.802 Kb size after restriction with NcoI and Pml1
Insert: HCC-scFv- the size of the insert DNA was 0.946 Kb which was restricted with
NcoI and Pml1 restriction enzyme. The concentration of the insert to be taken was
calculated using the Equation (4.5.2.1) and it was found to be 28.95ng. The insert
DNA was taken in 3 moles excess of vector DNA.
4. The controls used for ligation setup were carried out as in Table 4.5.2.2.
xciv
Content DNA Water,
nuclease-
free
5X Rapid
Ligation
Buffer
T4 DNA
ligase(5 unit/
µl)
Uncut pCAMBIA
1301
2 µl(100ng) 18 µl
Cut
pCAMBIA1301
2 µl(100ng) 18 µl
Cut (pCAMBIA
1301)+ligated
2 µl(100ng) 13 µl 4 µl 1 µl
Experiment-1
Vector(cut-9.802 kb)
+
Insert(0.946 kb)
2 µl(100ng)
1 µl(29ng)
12 µl
4 µl
1µl
Experiment-2
Vector(cut-9.802 kb)
+
Insert(0.946 kb)
2 µl(100ng)
2 µl(58ng)
11 µl
4 µl
1 µl
Table 4.5.2.2: Controls for Ligation setup.
5. The contents were vortexed and spinned briefly to collect the drops.
6. The mixture was incubated at 22°C for 5 min.
7. Ligation mixture of 2-5 µl was used for transformation.
The reaction mixture can be stored at 0°C to 4°C until used for transformation.
4.5.2.4.3. TRANSFORMATION OF THE LIGATED SAMPLES
4.5.2.4.3.1. COMPETENT CELL PREPARATION
A single colony of E. coli (DH5α) was inoculated into 2 ml of LB culture
broth and incubated (Scigenics, India) at 37°C for 10 hours to 12 hours overnight.
From the overnight culture, 500µl was inoculated into 50 ml of 2X YT medium and
incubated at 37°C in a rotary shaker until it reached an O.D of 0.5 to 0.6 at 600nm
(Biophotometer, Germany).To a sterile 50 ml polypropylene tube, 30 ml of the culture
xcv
was transferred and incubated in ice for 30 minutes. The culture was centrifuged at
5,000 rpm at 4°C for 10 minutes. The supernatant was discarded and the pellet was
resuspended in a small volume of medium left behind and finally the pellet was gently
resuspended in 30 ml of ice cold 100mM CaCl2 and incubated in ice for 30 minutes.
The resuspended cells were centrifuged at 5,000 rpm for 10 minutes at 4°C.The
supernatant was discarded and the pellet was resuspended gently in 3ml (1/10th
volume
of CaCl2) of ice cold 100mM CaCl2.The competent cells were stored in ice for atleast
30 minutes before use.
4.5.2.4.3.2. TRANSFORMATION OF COMPETENT CELLS (E. coli)
Competent cells of 200 µl was aliquated into 2ml sterile eppendorf tubes and
incubated in ice (cut tips were used to aliquot the cells). The liagted samples were
dispensed into the above eppendorf tubes containing competent cells separately and
incubated in the ice for 30 minutes. Heat shock was given for 2 minutes at 42°C in
water bath and then transferred and incubated in ice for 10 minutes.The cells were
transferred to 0.8 ml of prewarmed LB mix and incubated at 37°C in a rotary shaker
for 1 hour at 220rpm.The LB mixture was then centrifuged at 10,000 rpm for 5
minutes and the supernatant was discarded. From the pellet, 50 µl and 200µl of cells
were plated onto LB agar plate containing antibiotics kanamycin (50µg/ml). The plates
were incubated overnight at 37°C. The controls kept for the cloning experiment are
shown below in the Table 4.5.2.3.
S.No Innoculum Medium Volume to be
plated(µl)
xcvi
1.
2.
3.
4.
5.
Competent cells
Uncut plasmid vector
Vector cut
Vector cut+ ligated
Digested vector + insert +
ligase(Experiment:1&2)
(a) LB [plain]
(b) LB+antibiotic
LB+antibiotic
LB+antibiotic
LB+antibiotic
LB+antibiotic
100
100
100
100
100
50 µl
200 µl
Table 4.5.2.3: Controls kept for the cloning experiment.
4.5.2.4.3.3. CONFIRMATION OF THE LIGATED CLONE
From the patched colony of clone, plasmid DNA was extracted miniprep
(chapter 4.4) and digested with restriction enzyme (chapter 4.5.1). The digested
samples were seperated in 0.8% agarose gel and the clones were confirmed using 1 kb
ladder.
4.6. TRANSFORMATION OF pCAMBIA 1301-HCC-scfv FROM E. coli DH5α
INTO Agrobacterium tumefaciens EHA105
xcvii
4.6.1. OBJECTIVE
To transform pCAMBIA 1301-HCC-scFV from E. coli DH5α into
Agrobacterium tumefaciens EHA105 by triparental mating.
4.6.2. PRINCIPLE
The gene of interest can be transformed into Agrobacterium tumefaciens by
electroporation, free/thaw transformation and triparental mating (Figure 4.6.1).
Triparental mating is an effective method to transfer a non-conjugative plasmid but
mobilizable plasmid into Agrobacterium. In this method two E. coli strains are used to
transfer the plasmid into Agrobacterium. E. coli strain (helper strain) carrying a
conjugative plasmid (helper plasmid), which encodes the gene of all the proteins for
the formation of mating bridge, to transfer itself or mobilize another plasmid into a
recipient cell. The helper plasmid contains mob (mobilization) and tra (transfer
functions) genes. The donar plasmid (cloning vector) contains oriT (specific origin of
transfer) and bom (activation site at which mob and tra gene product can act). The
plasmid from the E. coli (donar strain) containing the gene of interest to be
transformed into Agrobacterium is transferred by the trans-acting elements in the
helper plasmid. The trans-acting functions include single stranded break within the ori
T region and target the nicked DNA (relaxosomes) into the mating bridge that links
donor and the recipient cells(Waters 1999).
The interaction between oriT and transfer functions are specific (Waters
1999, Hamilton 2000).Widely used helper plasmid is pRK2013 which carries the
transfer and mobilization functions of RK2 on a colE1 replicon (Ditta 1985). Thus
xcviii
pRK2013 can transfer plasmid carrying RK2 derived oriT from E. coli into
Agrobacterium tumefaciens. Transfer of T-DNA from E. coli into Agrobacterium by
triparental mating has been described by Roger et al (2000). After mating
Agrobacterium strains harboring the transformed vector (Ti plasmid containing gene
of interest) are selected on antibiotic plates depending on the antibiotic resistant
marker present on the plasmid.
Figure 4.6.1: Illustration of transformation of pCAMBIA 1301-HCC-scFv into
A.tumefaciens EHA 105 by triparental mating
xcix
4.6.3. MATERIALS
Recipient strain – Agrobacterium tumefaciens EHA 105 (super-virulent strain)-
obtained from Dr. Tomasz Pniewski, (Institute of Plant Genetics, Polish Academy of
Sciences, Poland) – Appendix 2.
Strain grows at 30°C on AB minimal medium with relevant antibiotics.
Donor strain - E. coli DH5α (containing transformed pCAMBIA 1301-ScFv)-the
plasmid to be mobilized. The strain grows at 37°C on LB medium with relevant
antibiotics (Kanamycin).
Helper strain - E. coli HB101 (containing pRK2013)-MTCC -398 (IMTEC, India)-
This plasmid helps the mobilization of the plasmid from donor to recipient strain. The
strain grows at 37°C on LB medium containing the relevant antibiotic (Kanamycin or
ampicillin)
LB medium (1000 ml) pH 7.2- (Kanamycin-50 µg/ml- E. coli DH5α, E. coli HB101)
Tryptone 10.0g
Yeast Extract 5.0g
Nacl 10.0g
Agar (1.5%) 15.0g
YEP medium (50ml) pH 7.1
Yeast Extract 1% 0.5g
Peptone 1% 0.5g
NaCl 0.5% 0.26g
Agar 1.6% 0.8g
c
AB Buffer (20X): pH 7.0 100ml
K2HPO4 (anhydrous) -6.0g
NaH2PO4 (anhydrous) -2.0g
Or
NaH2PO4 .2H2O (hydrated)-2.6g
Each salt was dissolved separately in about 50 ml of autoclaved Millipore water and
made up to 100ml. The pH was adjusted to 7.0. The buffer was autoclaved and stored
at room temperature.
AB salts (20X) -100ml
NH4cl - 2.0g
MgSo4.7H2O - 0.6g
Kcl - 0.3g
Cacl2.2H2O - 0.31g
FeSO4.7H2O - 0.005g
All the salts were dissolved in 80ml of Millipore water and made up to 100ml. The
contents were autoclaved and stored at room temperature.
AB minimal medium (Agrobacterium tumefaciens EHA 105)
For pour plate inoculation of the serially diluted samples, 400 ml of AB minimal
medium was prepared by adding 2 g glucose and 6 g agar to 360 ml of millipore water
and autoclaved. To this 20 ml of AB salt and 20 ml AB buffer was added aseptically
and respective amount of antibiotics, rifampicin and kanamycin were added.
For streaking of the colonies obtained from serial dilution, 300ml of AB minimal
medium was prepared by adding 1.5 g glucose and 4.5 g Agar to 270 ml of millipore
water and autoclaved. To this 15 ml of AB salt and 15 ml of AB buffer was added
ci
aseptically and respective amount of antibiotics from rifampicin and kanamycin stock
were added.
Antibiotic stocks
Stock: Rifampicin (10mg/ml)
Working concentration: Rifampicin (10µg/ml)
Stock: Kanamycin (100mg/ml)
Working concentration: Kanamycin (100µg/ml, 50µg/ml)
4.6.4. METHOD
The day on which triparental mating is performed was considered as day
one.
Day: -4
Agrobacterium tumefaciens (EHA 105) was streaked to get single colonies on AB
minimal medium agar or YEP agar plates containing antibiotic (rifampicin-10µg/ml)
and incubated at 30°C.
Day: -1
E. coli HB101 (containing pRK2013) was streaked to get single colonies on LB agar
with 50µg/ml of kanamycin and incubated at 37°C.
E. coli DH5α (containing transformed pCAMBIA 1301-ScFv) was also streaked on
LB agar with 50µg/ml of kanamycin and incubated at 37°C.
Day: +1
A plate with YEP agar plate was prepared without antibiotics. One colony of E. coli
DH5α(pCAMBIA 1301-HCC-scFv) and E. coli HB101(pRK2013) were spotted over
YEP agar medium separately and 3 closely lying colonies of A. tumefaciens (EHA
cii
105) were picked and spotted near to the other 2 strains of E. coli using a sterile loop,
all three bacterial strains were mixed well. Then the plates were incubated at 30°C for
12 to 18 hours.
Day: +2
The colony obtained as a result of mating was scrapped and suspended in 1.0 ml of
0.9% sterile sodium chloride solution. Serial dilution was performed by transferring
0.1 ml of bacterial suspension into 0.9% NaCl. Similarly six dilutions were made up to
10-6
. 100 µl of each dilution was plated on AB minimal medium containing
Rifampicin (10µg/ml) and Kanamycin (50µg/ml). The cultures were uniformly spread
onto the surface of the media using L rod by spread plate technique. The plates were
incubated at 30°C for 3 to 5 days.
*Note: Before each transfer the contents were vortexed thoroughly.
Day: +6
At one or two dilutions, single colonies appear on AB minimal medium. These
colonies are Agrobacterium tumefaciens into which the donor plasmid has been
transformed.
Day: +7
Six to eight single colonies of Agrobacterium tumefaciens were then streaked on AB
minimal medium containing antibiotics and incubated at 30°C for 2 to 3 days to get
single colonies.
Day: +11
Then the single colonies were patched and maintained as master plate for further use in
duplicates.
ciii
A glycerol stock of the transconjugants was made immediately and used for further
studies.
4.7. CALLUS INDUCTION OF IR64 RICE VARIETY FOR
TRANSFORMATION
4.7.1. OBJECTIVE
To induce callus by indirect organogenesis in IR64 rice variety for
Agrobacterium mediated transformation
4.7.2. PRINCIPLE
Plant tissue culture is a technique whereby the explants are isolated from
the plant and grown aseptically on a nutrient medium under controlled environmental
conditions (16/8 hours of light/dark and 23ºC to 25ºC)
Organogenesis is the formation of either shoots or roots and depends on the
balance between the auxin and cytokinin phytohormones during invitro culture.
Organogenesis is of two types indirect and direct. During indirect organogenesis, the
organs are formed indirectly via a callus phase and in direct organogenesis, the organs
are formed without undergoing callus phase. Induction of plants via callus phase has
been widely used for the production of transgenic plants.
civ
Totipotency is the ability of a single plant cell to regenerate into an entire
plant. Callus is undifferentiated mass of parenchymatous cells. Callus induction from
the embryo is very useful to obtain an increased number of hybrid plants. Formation of
shoots or roots from a callus is known as organogenesis. First report on organogenesis
was made (White 1939). Induction of callus shoots and roots can be done from any
part of the plant using different medium. The two major classes of growth regulators
essential for both callus induction and organogenesis are auxins and cytokinins.
Auxin induces cell division, cell elongation, swelling of tissues and the
formation of adventitious roots. At low concentration of auxin, adventitious root
formation takes place, whereas at high concentration of auxin root formation fails to
occur and callus formation takes place. Cytokinins are derivatives of adenine and have
important role in shoot induction. They usually promote cell division if added together
with an auxin. At higher concentration (1 to 10 mg/L), adventitious shoot formation is
induced but root formation is generally inhibited.
In this experiment 2,4D was used for callus induction and later for
regeneration NAA (Naphthalene acetic acid) and kinetin was used as auxin and
cytokinin respectively.
4.7.3. MATERIALS
1. Explant: Healthy matured Rice (Oryza sativa L) seeds of IR64 (Indica rice variety
64) was obtained from ICAR, Cuttack, India.
2. Sterilizing Solutions: 70% ethanol, 3-4% sodium hypochloride (Merck) with 1-2
drops of Tween-20(Sigma), 0.2 % (w/v) HgCl2
(mercuric chloride) (Rankem), sterile
millipore water.
cv
3. Stock solution: 2, 4-Dichlorophenoxy acetic acid (2, 4-D; Sigma)
20mg/100ml.Dissolve the flake in a few drops of 0.1 N KOH and make the volume to
100ml with autoclaved millipore water and store at 4°C.
4. MS 2, 4-D medium: MS salts(Merck) and vitamins (Sigma), 300mg/L casamino
acid, 2mg/L 2, 4-D and 8 g/L agar, pH 5.8 (Autoclaved) (Table 4.7.1).
Name of
stock
solution
Chemicals
1000ml
500ml
250ml 1Quantit
y
g/L
*V of
stock
1Quantit
y
g/L
*V of
stock
1Quantit
y
g/L
*V of
stock
MS 1 NH4NO3 82.5 20 41.25 10 20.625 5
KNO3 95.0 47.5 23.75
MS2 MgSO4.7H2O 37.0 10 18.5 5 9.25 2.5
MnSO4.4H2O 2.23 1.115 0.5575
ZnSO4 1.058 0.529 0.2645
CuSO4.5H2O 0.0025 0.00125 0.000625
MS3 Cacl2.2H20 44.0 10 22.0 5 11.0 2.5
KI 0.083 0.0415 0.02075
CoCl2.6H2O 0.0025 0.00125 0.000625
MS4 KH2PO4 17.0 10 8.5 5 4.25 2.5
H3BO3 0.62 0.31 0.155
Na2MoO4.2H2
O
0.025 0.0125 0.00625
MS5 FeSO4.7H2O 2.785 10 1.3925 5 0.69625 2.5
Na2EDTA.2H2
O
3.725 1.8625 0.93125
Vitamins Nicotinic acid 10mg/10
0ml
5 10mg/10
0ml
2.5 10mg/10
0ml
1.25
Pyridoxine
HCl
10mg/10
0ml
10mg/10
0ml
10mg/10
0ml
Thiamine HCl 20mg/10
0ml
20mg/10
0ml
20mg/10
0ml
Glycine 40mg/10
0ml
40mg/10
0ml
40mg/10
0ml
Hormone 2,4D 10mg/10 20 10mg/10 10 10mg/10 5
cvi
0ml 0ml 0ml
Myoinositol 0.1g 0.05g 0.025g
Casamino
acid
0.3g 0.15g 0.075g
Sucrose/
Maltose
30g 15g 7.5g
Agar 8.0 4g 2g
Table 4.7.1: Chemical composition of MS 2, 4-D medium
*V- Volume of stock to prepare the media 1Quantity- quantity of chemicals required to prepare the stock
4.7.4. METHODS
1. The seeds of IR 64 were manually dehusked.
2. The dehusked seeds were surface sterilized with 70% (v/v) ethanol for 1 min and
then with 0.2 % (w/v) HgCl2 (mercuric chloride) for 10 minutes with gentle agitation
and rinsed several time with sterile Millipore water to remove HgCl2. It was further
sterilized with 3 to 4% sodium hypo chloride (containing 2 to 3 drops of tween 20) for
30 minutes with gentle agitation and rinsed with autoclaved sterile Millipore water.
3. Individually twenty five embryos from the sterilized seeds were placed in each Petri
dish containing 20 ml of MS 2, 4-D medium.
4. The plates were incubated in the dark at 25 ± 1ºC.
5. After 3 to 4 days, the emerging shoots and roots were cut. The remaining scutellar
tissues were subcultured onto a fresh medium with the same orientation.
cvii
6. Yellowish white soft embryogenic calli developed on the surface of the scutellar
tissue after 2 to 3 weeks.
7. Callus (total/embryogenic) induction frequency (%) can be calculated as in Equation
(4.7.1).
4.8. TRANSFORMATION OF pCAMBIA 1301-HCC-scFv FROM
Agrobacterium tumefaciens EHA 105 INTO CALLUS BY Agrobacterium
MEDIATED TRANSFORMATION
4.8.1. OBJECTIVE
To transform pCAMBIA 1301-HCC-scFv gene from Agrobacterium
tumefaciens EHA 105 into callus by Agrobacterium- mediated transformation.
4.8.2. PRINCIPLE
The Plant pathogenic Agrobacterium tumefaciens have the capacity to
transfer part of its T-DNA into the nuclear genome of the plant cells. Two types of
Agrobacterium strains that are used for plant genetic transformation are A. tumefaciens
cviii
and A. rhizogenes. In case of A. tumefaciens strains, the oncogenes are encoded in the
T-DNA (Ti) which will induce tumor formation on the infected plant tissue and in case
of A. rhizogenes strains, the T-DNA (Ri) genes encode oncogenes that will induce the
production of adventitious roots called the hairy root tissue. The transfer of T-DNA
depends on the expression of the vir genes which transfers the DNA into plant nucleus
by recognizing specific sequence called right and left border (RB and LB) sequence.
Several strategies to introduce foreign genes directly into the T-region of Ti
plasmids have been developed by Hoekema et al (1983) and de Frammond et al
(1983). A binary vector system was developed were the replicon harboring the T-DNA
region constituted the binary vector, whereas the replicon containing the vir genes was
known as vir helper. The vir helper plasmid does not induce tumor but contain
complete or partial T-region. A number of Agrobacterium strains containing non-
oncogenic vir helper plasmids have been developed, including LBA4404 (Ooms
1981), GV3101 MP90 (Koncz 1986), AGL0 (Lazo 1991), EHA101 and its derivative
strain EHA105 (Hood 1986, 1993) and NT1 (pKPSF2) (Palanichelvam 2000).
Commonly used non- oncogenic A. tumefaciens vectors have the oncogenic
portion of the T-DNA replaced by a dominant selectable marker gene. In this
experiment A. tumefaciens EHA 105 transformed (Karabi Datta and Swapan Datta,
2006) with pCAMBIA 1301-HCC-scFv was allowed to infect the callus of IR 64 (
Figure 4.8.1) .The selectable marker gene present in pCAMBIA 1301 is hpt gene
which codes for hygromycin phosphotransferase which detoxifies the aminocyclitol
antibiotic hygromycin B. Most of the cereals exhibit higher sensitivity to hygromycin
B than to kanamycin. So this gene can be used to screen the transformed callus. The
reporter gene gus A was not used as it was excised and inserted with the gene of
interest.
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Figure 4.8.1: Illustration of transformation of pCAMBIA 1301-HCC-scFv into IR
64.
4.8.3. MATERIALS
AB Buffer (20X): pH 7.0 100ml
K2HPO4 (anhydrous) -6.0g
NaH2PO4 (anhydrous) -2.0g
Or
NaH2PO4 2H2O (hydrated)-2.6g
Each salt was dissolved separately in about 50 ml of autoclaved Millipore water and
made up to 100ml. The pH was adjusted to 7.0. The buffer was autoclaved and stored
at room temperature.
AB salts (20X) -100ml
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NH4cl - 2.0g
MgSo4.7H2O - 0.6g
Kcl - 0.3g
Cacl2.2H2O - 0.31g
FeSO4.7H2O - 0.005g
All the salts were dissolved in 80ml of Millipore water and made up to 100ml. The
contents were autoclaved and stored at room temperature.
AB minimal medium (Agrobacterium tumefaciens EHA 105)
For pour plate inoculation of the serially diluted samples, 400 ml of AB minimal
medium was prepared by adding 2 g glucose and 6 g agar to 360 ml of millipore water
and autoclaved. To this 20 ml of AB salt and 20 ml AB buffer was added aseptically
and respective amount of antibiotics rifampicin and kanamycin were added.
For streaking of the colonies obtained from serial dilution, 300ml of AB minimal
medium was prepared by adding 1.5 g glucose and 4.5 g Agar to 270 ml of millipore
water and autoclaved. To this 15 ml of AB salt and 15 ml of AB buffer was added
aseptically and respective amount of antibiotics from rifampicin and kanamycin stock
were added.
Antibiotics
Stock: Rifampicin (10mg/ml)
Working concentration: Rifampicin (20µg/ml)
Stock: Kanamycin (100mg/ml)
Working concentration: Kanamycin (50µg/ml)
Stock: Cefotaxime (Invitrogen):100mg/ml. Filter sterilize. Store at -20°C.
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Working concentration (250mg/L)
Selective marker agent
Hygromycin B: (H3274-50MG- Sigma)
10mM MgSO4
10mM MgSO4 was mixed in water and sterilized by autoclaving and stored at -20°C.
Acetosyringone (3, 5, dimethoxy 4 hydroxy-acetophenone) (Sigma-Aldrich)
40mg/ml 200mM): The powder was dissolved in DMSO (dimethyl sulfoxide, Sigma)
and the solutions were made up using autoclaved Millipore water. The solution was
filter sterilized and stored at 4°C.
Kinetin (6 furfurylaminopurine) (Sigma):20mg/ml. The powder was dissolved in a
few drops of 1N HCl, and then the volume was made up with autoclaved Millipore
water and stored at 4°C.
Components Quantity(mg/L)
1. Macronutrients
Cacl2.2H2O
MgSO4.7H2O
NaH2PO4.H2O
KCl
150.0
250.0
150.0
2950.0
2 Micronutrients
KI
H3BO3
MnSO4.H2O
ZnSO4.7H2O
Na2MoO4.2H2O
CuSO4.5H2O
0.75
3.0
10.0
2.0
0.25
0.025
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CoCl2.6H2O 0.025
3 Iron composition
Na2EDTA
FeSo4.7H2O
Vitamins
Nicotinic acid
Pyridoxine HCl
Thiamine HCl
Glycine
Inositol
37.3
27.8
0.5
0.5
1.0
2.0
100.0
4 L-glutamine
Aspartic acid
Arginine
Casamino acid
Sucrose
Glucose
Acetosyringone(3,5,dimethoxy 4 hydroxy-
acetophenone)
876.0
266.0
174.0
500.0
68.5g/L
36g/L
200µM
Table 4.8.1: Chemical composition of AAM Medium
The components were mixed and then pH was adjusted to 5.2. The medium was
sterilized by filter sterilization (Hiei 1994).
Name of stock
solution
Components Quantity
g/L
Volume of
stock for
1L(ml)
Final
concentration
(mg/L)
N61
KNO3 141.50 20 2830.0
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N62 MgSO4.7H2O
MnSO4.4H2O
ZnSO4
(NH4)2SO4
18.5
0.44
0.15
46.3
10 185.0
4.4
1.5
463.0
N63 Cacl2.2H20 16.6 10 166.0
N64 KI
KH2PO4
H3BO3
0.08
40
0.16
10 0.8
400
1.6
N65 FeSO4.7H2O
Na2EDTA.2H2O
2.785
3.725
10 27.85
37.25
Vitamins Nicotinic acid
Pyridoxine HCl
Thiamine HCl
Glycine
10mg/100ml
10mg/100ml
20mg/100ml
40mg/100ml
5 0.5
0.5
1.0
2.0
Hormones
Myoinositol
Sucrose/maltose
Agar
2,4 D 10mg/100ml 20 10.0
100.0
30,000.0
8000.0
Table 4.8.2: Chemical composition of N6 Medium
The pH of the N6 Medium was adjusted to 5.6-5.8. All the chemicals were obtained
from Merck. Vitamins and hormones were obtained from Sigma (Karabi Datta, 2006)
N6-AS medium for infiltration (liquid) and co-cultivation (solid)
N6 salts from the N6 salts Table 4.8.2, MS vitamins from the MS Table 4.7.1,
300mg/L casamino acid, 2mg/L2, 4-D, 30 g/L sucrose and 10 g/L glucose. In case of
cocultivation medium 9 g/L agar was added. The pH was adjusted to 5.2. The medium
was autoclaved and cooled to 55°C before adding acetosyringone (200µM).
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NAA (1-Naphthylacetic acid-Sigma):20mg/ml. the powder was dissolved in a few
drops of 0.1N NaOH and finally made up the volume with autoclaved Millipore water
and stored at 4°C.
Selection Medium
MS salts, MS vitamins, 300mg/L casamino acid, 2mg/L 2, 4-D, 250mg/L cefotaxime
(added after autoclaving), 8g/L agar. The pH was adjusted to 5.8.
For hpt gene selection: to the above selection medium 30g/L sucrose was added and
autoclaved. After autoclaving the medium was cooled to 60 °C and then 50 mg/L
hygromycin was added.
Regeneration medium (MSKN)
MS salts, MS vitamins, 2mg/L kinetin, 1mg/L NAA, 300mg/L casamino acid, 50mg/L
cefotaxime, 30g/L sucrose, 10g/L sorbitol, 2.5g/L gelrite. The pH was adjusted to 5.8
and the medium was autoclaved.
Rooting medium (MSO)
MS salts, MS vitamins, 30g/L sucrose, 2.5g/L gelrite. The pH was adjusted to 5.8 and
the medium was autoclaved.
4.8.4. METHOD
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Preculturing of Agrobacterium strain
Day 1: A loop full culture was taken from a glycerol stock culture of
Agrobacterium transformed with pCAMBIA 1301-HCC-scFv and streaked
on to AB minimal medium containing 20 mg/ml rifampicin and 50mg/ml
kanamycin.
The plates were incubated in the dark at 28°C for 2 days.
Preculturing of calli
Day1: Embryogenic calli of 1.3-3mm diameter size calli was selected from
4 to 5 weeks old culture and transferred into fresh MS 2, 4 D medium
(approx 50 Calli /dish).
The plates were incubated at 28°C in the dark for 3 days.
Culturing of Agrobacterium strain
Day 3: Three to four well grown Agrobacterium colonies were transferred to
50 ml of AAM medium (Table 4.8.1) containing 20mg/ml of rifampicin and
50 mg/ml of Kanamycin in a 250ml conical flask
The conical flask was kept in a shaker at 250 rpm for 20 to 30 hours at
28°C.
Transformation
1. Day 4: Acetosyringone of 200 µM was added to the Agrobacterium suspension
and kept in shaker at 250 rpm for 2 hours at 28°C.
2. The bacterial culture was transferred into a 50 ml centrifuge tube and
centrifuged at 3,500g for 30 minutes at 10°C.
3. To the pellet 20 ml of Mg So4 was added and the OD measurement of the
Agrobacterium suspension was measured at 600nm.
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4. The above bacterial suspension was centrifuged at 3,500g for 30 minutes at
10°C and the supernatant was discarded.
5. The pellet was resuspended in a small volume of liquid N6-AS in filtration
medium by pipetting up and down.
6. The OD of the final volume of agrobacterial suspension was adjusted to 1 with
N6-AS medium.
7. The bacterial suspension was transferred into a sterile Petri plate and then the
precultured embryogenic calli was put into the bacterial suspension.
8. The Petri plate was placed open in a vacuum desiccator for 10 minutes.
9. After another 20 minutes, the bacterial suspension was pipetted out from the
embrogenic calli.
10. Excess Agrobacterium-infection medium was absorbed from the calli using
sterile filter papers.
11. The calli was then transferred to N6-AS solid co-cultivation medium and the
plates were incubated at 28°C for 3 days under dark condition.
Calli Proliferation
After 3 days, the calli was transferred on MS-2, 4-D medium with 250mg/L
cefotaxime for proliferation of the calli.
The calli was incubated in the dark at 25°C for 10 days.
Selection
1. The calli was first selected on a medium containing antibiotic (250mg/l
cefotaxime) and selective agent (50mg/ml hygromycin B) and kept in the dark
for 2 weeks at 25 °C.
2. The survining healthy embryogenic calli was transferred from the first selection
media into the second selection media containing antibiotic (250mg/l
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cefotaxime) and selective agent (50mg/ml hygromycin B) and kept in the dark
for 2 weeks at 25°C.
3. The survining embryogenic calli was further subcultured on a third selection
medium containing antibiotic (250mg/l cefotaxime) and selective agent
(50mg/ml hygromycin) and kept in the dark for 2 weeks at 25 °C.
Regeneration
1. The healthy surviving embryogenic calli was transferred into MSKN
regeneration medium and the cultures were kept in the dark for 20 days.
2. The emerging shoots were harvested and transferred again to a fresh
regeneration medium (MSKN) in a Petri dish. The Petri dish was placed in the
light at 27°C with 16 hours photoperiod (110µmol/m2/s) for 5 days.
3. The shoots which grew well were transferred into 25 ml conical flask
containing MSKN regeneration medium. The flask was placed in the light at
27°C with 16 hours photoperiod (110µmol/m2/s) for 10 to 20 days.
4. The healthy shoots were transferred into the rooting medium (MSO).
5. The percentage (%) of callus regeneration frequency (CRF) was calculated as
Equation (4.8.1)
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4.9. EXTRACTION OF TOTAL GENOMIC DNA FROM PLANT TISSUES
FOR MOLECULAR ANALYSIS
4.9.1. OBJECTIVE
The plant genomic DNA was isolated by CTAB method which can be used
for molecular analysis to confirm the presence of the transformed gene.
4.9.2. PRINCIPLE
The extraction of genomic DNA from plant tissues requires the digestion of
cell wall to release the cellular components (Rogers 1994). The nucleases released
from the cellular components had to be inactivated and the genomic DNA has to be
separated from the cellular debris. The cellular contents are released by grinding the
tissue in liquid nitrogen with a motor and pestle. The liquid nitrogen also prevents
nuclease activity. The plant tissue contain large amount of carbohydrates and therefore
DNA is isolated using either SDS or CTAB (Cetyltrimethylammonium bromide).
CTAB protocol was developed by Murray and Thompson (1980) which is used for the
extraction and purification of DNA from plants and it is suitable for the elimination of
polysaccharides and polyphenolic compounds which affect the DNA purity and
quality.
The plant cells are lysed with the ionic detergent CTAB. The CTAB forms
stable and soluble complex with DNA (CTAB-DNA complex) under high salt
concentration. When the salt concentration was reduced to below 0.4 M NaCl causes
cxix
the CTAB-nucleic acid complex to precipitate leaving the polysaccharides, phenolics
and other contaminants in the supernatant. Thus the extraction buffer with 2% CTAB
takes care of the release of genomic DNA from the cell content. During extraction
different concentration of CTAB is used which helps in the removal of
polysaccharides. Addition of chloroform: isoamyl alcohol denatures and degrades
protein and helps in the removal of CTAB. NaCl concentration in CTAB solutions is
reduced in successive steps, which aid in DNA precipitation by salting out principle.
EDTA and PVP act as chelating agents. They prevent binding of phenolic compounds
to DNA. Mg+ is a cofactor for nucleases. EDTA binds to Mg+ and prevents nuclease
activity. High salt TE dissolves DNA and CTAB. In 95% ethanol precipitation, DNA
does not dissolve in ethanol and gets precipitated but CTAB gets dissolved and is
removed with the supernantant. 70% ethanol washes and removes the traces of salt and
CTAB.
The isolated genomic DNA was further purified using the PCR clean up kit
and then the amount of DNA was estimated using Biophotometer. DNA had maximum
absorption at 260nm. Optical density of 1 at 260nm corresponds to 50 µg/ml of double
stranded DNA. The ration of O.D 260/280 provides the purity of the DNA. A pure
DNA will have a ratio of 1.8. Ratio less than 1.8 indicates probably the presence of
proteins or other UV absorption. Ratio higher than 2.0 indicates that the sample may
be contaminated with chloroform or phenol and should be precipitated again with
ethanol.
4.9.3. MATERIALS
DNA EXTARCTION
2X CTAB buffer (100ml)
2%CTAB (Cetyltrimethylammonium bromide) (w/v)-(Sigma)
100mM Tris, pH 8.0
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20mM EDTA, pH8.0
1.4 M NaCl
1% PVP
CTAB of 2g was dissolved in 70 ml of water. NaCl of 8.18g was added and dissolved.
To the above 10ml of Tris (1M, pH8.0) and 4ml of EDTA (0.5 M, pH 8.0) was added.
The final volume was made up to 99ml with autoclaved Millipore water. The buffer
was autoclaved and then 1g of PVP was added and dissolved.
5% CTAB (25ml)
5% CTAB
0.35M NaCl
CTAB of 1.25g was dissolved in 20 ml autoclaved Millipore water and then 0.5g of
NaCl was added and dissolved. The final volume was made up to 25ml with
autoclaved Millipore water and autoclaved.
CTAB precipitation buffer (100ml)
1%CTAB
50mM Tris, pH 8.0
10mM EDTA, pH8.0
CTAB of 1 g was dissolved in 90ml of autoclaved Millipore water and then 5ml of
Tris (1M, pH8.0) and 2ml of EDTA (0.5 M, pH 8.0) was added. The final volume was
made up to 100ml and autoclaved.
High salt TE buffer (50ml)
10 mM Tris, pH 8.0
1.0 mM EDTA, pH8.0
1 M NaCl
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NaCl of 2.922g was dissolved in 40 ml of Millipore water. To this 500 l of Tris (1M,
pH 8.0) and 100 l of EDTA (0.5 M, pH 8.0) was added and the volume was made up
to 50 ml and autoclaved.
0.1X TE (100ml)
1mM Tris pH8.0
0.1mM EDTA pH8.0
100 l of Tris (1M, pH 8.0) and 50 l of EDTA (0.5 M, pH 8.0) was added and the final
volume was made up to 100ml with Millipore water and autoclaved.
PURIFICATION OF DNA FOR PCR
Gel elution Kit (Real Genomics, Hi Yield TM
Gel/PCR DNA MINI KIT-
YDF100/YDF300)
100 mini preps/kit
DF Buffer: 80ml
W1 Buffer: 45ml
Wash Buffer (concentrated):25ml*
Elution Buffer: 6ml
DF Column: 100pcs
2ml Collection Tube: 100pcs
*Initially 100ml of ethanol (96-100%) was added to 25 ml of Wash buffer prior to use
1.5 ml eppendorf tubes
DNase-free pipette tips
4.9.4. METHOD
4.9.4.1. DNA EXTRACTION
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1. To one gram of the plant material, liquid nitrogen was added and grinded into fine
powder with chilled sterile motor and pestle.
2. Approximately 100mg of the grinded material was transferred into chilled 2ml
microfuge tubes. The content was transferred using chilled spatula and 500 µl of
hot (65°C) CTAB extraction buffer was added.
3. The tubes were mixed well by inversion and equal volume (700 µl) of ice cold
chloroform: isoamyl alcohol (24:1) was added.
4. The content was mixed well by inversion to form an emulsion and centrifuged for
10 minutes at 13,000rpm in a micofuge. The top aqueous phase was transferred
into new microfuge tubes using cut tips.
5. The volume was measured and 1/5th
volume of 5%CTAB solution was added and
mixed well by gently inverting the tubes.
6. Equal volume of chloroform: isoamyl alcohol (24:1) was added and mixed
thoroughly to form an emulsion and centrifuged at 13,000 rpm for 10 minutes.
7. The aqueous phase was transferred into fresh 1.5ml microfuge tubes using cut tips
and equal volume of CTAB precipitation buffer was added and mixed gently by
inversion.
8. The tubes were centrifuged at 10,000rpm for 1 minute. The supernatant was
discarded and the pellet was dissolved in 200 µl of high salt TE buffer by gently
tapping the tube.
9. If the DNA was not dissolved, the tubes were kept in water bath at 65 °C for 10
minutes and the content was centrifuged at 13,000rpm for 10 minutes and the
supernatant was transferred into a fresh microfuge tube. High salt TE buffer of 100
µl was added again, if the pellet was not dissolved. Then the contents were
centrifuged at 13,000 for 10 minutes and the supernatant were pooled together.
10. To the supernatant 2.5 volumes of ice cold 95% ethanol was added to the solution
and mixed gently by inversion and centrifuged for 15minutes at 13,000rpm at 4°C.
cxxiii
11. The supernatant was discarded and to the pellet 70% ethanol was added up to the
original volume and centrifuged at 13,000rpm for 10 minutes at 4°C.
12. The supernatant was discarded and the pellet was dried. The pellet was then
dissolved in 50 µl of 0.1X TE or sterile water.
4.9.4.2. PURIFICATION OF DNA FOR PCR
Sample preparation
Extracted DNA of 100 µl was transferred into 1.5 ml microcentrifuge tube.
DF buffer of 5 volumes were added to 1 volume of the sample and mixed by
vortexing.
DNA Binding
DF column was placed in a 2 ml collection tube.
The sample mixture from step 2 was added into DF Column.
The column was centrifuged at full speed approximately 13, 000 rpm for 30
seconds.
The flow through was discarded and the DF Column was placed back into the 2
ml collection Tube. When the sample was more, this step was repeated.
Wash
W1 Buffer of 600 µl was added into the DF Column and allowed to stand
for 1 minute.
The column was centrifuged at full speed approximately13, 000 rpm for 30
seconds.
cxxiv
The flow through was discarded and the DF column was placed back into
the 2 ml collection Tube.
The column was centrifuged at full speed approximately13, 000 rpm for 3
minutes to dry the column matrix.
DNA Elution
The dried DF column was transferred into a new 1.5ml microcentrifuge
tube.
Elution buffer or TE of 20 µl to 50 µl was added into the center of the
column matrix.
The elution buffer was allowed to stand for 2 minutes until the buffer is
absorbed by the matrix.
The column was centrifuged at full speed for 2 minutes to elute the purified
DNA
The eluted DNA of 2 µl was diluted with 48 µl of sterile water and the
quantity was measured using biophotometer.
4.9.4.3. ESTIMATION OF DNA
To measure the amount of DNA, the purified samples were diluted using
sterile water and measured using Biophotometer.Sterile water was used as blank.
The biophotometer displays the amount of DNA present in the samples
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4.10. MOLECULAR ANALYSIS OF PUTATIVE TRANSFORMED PLANTS
BY POLYMERASE CHAIN REACTION (PCR)
4.10.1. OBJECTIVE
Molecular analysis of transformed plant was carried out by detecting the
presence of CaMV35S promoter gene to confirm gene transformation.
4.10.2. PRINCIPLE
cxxvi
The most common strategy employed for screening transgene is PCR-based
detection of transgene followed by gel electrophoresis and compared with the markers.
PCR is a technique used to amplify a specific DNA sequence into millions of copies.
The enzyme Taq polymerase (94Kb) isolated from Thermus aquaticus is used in this
technique which is thermostable and resistant to denaturation by heat treatment and
active at 75°C to 80 °C. This enzyme is used to amplify minute quantities of transgene
DNA to a detectable level on 1% agarose gel. PCR based detection strategy is
extremely sensitive.
A primer is a short single stranded oligonucleotide which when attached to
a single stranded template molecule acts as a starting point for complementary strand
synthesis directed by DNA polymerase enzyme. PCR amplification involves two
oligonucleotide primers that flank the DNA sequence to be amplified. The primers
hybridize to the opposite strand of the target sequence such that the DNA synthesis by
the polymerase enzyme proceeds across the region between the primers effectively
doubling the amount of that DNA sequence. Since the extension products are also
complementary and capable of binding to the primers, the cycle is repeated after
denaturation step. By repeated cycles of denaturation, priming and extension there is a
rapid exponential accumulation of the specific target fragment. Thus after 10 cycles
the target sequence would be amplified 1000 folds.
Steps involved in PCR
The technique involves repeated cycles of DNA synthesis which is based on three
simple steps for amplification reactions (Mullis 1987). The three steps involved are
Denaturation of the template DNA into single strands
Annealing of primers
Extension of the new DNA strands
cxxvii
The three major steps are repeated for 30 to 40 cycles. This is carried out on an
automated thermo cycler, which can heat and cool the tubes containing the reaction
mixture in a very short time.
DNA Denaturation
During the denaturation, the double strand DNA is denatured into a single
stranded DNA and the enzymatic reaction are halted due to the high temperature. DNA
denaturation also eliminates the secondary structures which may interrupt DNA
amplification. This step is carried out from 94 C to 95 C for 1 minute. The
temperature depends upon various factors like nature of DNA, G-C content, secondary
structures etc. An initial denaturation of template DNA was carried out at 94 C to
95 C for 5 to 10 minutes for complete strand separation at the first PCR step.
Primer Annealing
The primers that are added to the reaction mixture keep moving due to
Brownian movement. Ionic bonds are constantly formed and broken between the
single stranded primer and the single stranded template. The stability of the bonds
increases when specific primers bind to the single stranded template and on this double
stranded region formed by the hybridization of the primer and specific nucleotide
sequence, the polymerase enzyme attaches and starts copying the template. Once few
bases are added to the template DNA, the ionic bond is so strong between the template
and the primer that it does not break. The annealing temperatures should be 5 C less
than the melting temperature Tm. The Tm of primers containing 18 to 24 base pairs
can be determined as in Equation (4.10.1). Higher annealing temperature result in no
amplification and lower annealing temperature results in non specific amplification.
Primer Extension
cxxviii
Primer extension results in synthesis of new strands usually carried out at
72 C. This is the ideal working temperature for the taq polymerase enzyme. The
primers, where the few bases are added to the strand, already have a strong ionic
attraction to the template DNA than the forces breaking these attractions. Primers that
are non specific for the template strand does not result in extension of the strand. For
every 1Kb target DNA, 1 minute is recommended.
b) Factors affecting amplification
Efficient amplification of target sequences depends on individual reaction compound,
time and temperature conditions of PCR reactions.
Sample volume
Sample volume of about 20 l, 50 l, 100 l volumes are recommended for PCR as it
assesses the adequate thermal equilibrium of reaction mixtures.
Template DNA
The purity and concentration of the template DNA affects the product yield
and the specificity of the PCR. Higher amount of template DNA may result in non
specific amplification or it will inhibit PCR amplification. A number of contaminants
such as SDS, phenol and other reagents used in template DNA preparation can inhibit
PCR.
Primers
It is one of the most important factors affecting the quality of PCR. The
primers usually used at equal concentration in the range of 0.1 to 1 M. Primers should
be free of complementary sequence at the 3’ end as it results in primer dimmer
formation which in turn reduces product yield. Typically 40 to 60 % of GC content is
recommended to avoid the formation of primer dimmer.
cxxix
Deoxy Nucleotide Tri Phosphates (dNTP’s)
Deoxy Nucleotide Tri Phosphate (dNTP) is the major source of phosphate
group in the reaction mixture. The final concentration of dNTP’s in the standard
amplification reaction mixture should be 200 M.
Taq DNA polymerase enzyme
Taq DNA polymerase lacks 3’5’ exonuclease activity but has 5’3’
exonuclease activity and 5’3’ polymerase activity. For most amplification 1.5– 2.0
units of enzyme is recommended. But higher enzyme concentration leads to non
specific amplification.
4.10.3. MATERIALS
The PCR ingredients and Primers were procured from Eppendorf, Germany and
Operon technologies, Germany. The sequence details of the primer for the detection of
CaMV35S promoter gene is provided below.
S.No. Primer Sequence
1
2
35s FP
35s RP
5’GCTCCTACAAATGCCATCA 3’
5’GATAGTGGGATTGTGCGTCA 3’
Sequence details of primers of CaMV35S
Negative control 1-Reaction mixture without the DNA sample
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Negative control 2- DNA isolated from regenerated plant from untransformed callus
Positive control- Restricted, eluted and purified pCAMBIA 1301(linearized plasmid-
eluted and purified using HiYield Gel/PCR DNA Mini kit (YDF100/YDF300)
4.10.4. METHOD
The following Reaction mixture was added in each tube in the same order
(Table 4.10.1).
S. No Reaction mixture Concentration Volume
1 10x Reaction buffer 1X 2.0 µl
2 MgCl2 5mM 2.0 µl
3 dNTP's 0.2mM 0.4 µl
4 Forward Primer 0.25mM 0.5 µl
5 Reverse Primer 0.25mM 0.5 µl
6 MilliQ water - 11.3 µl
7 DNA 20ng/ml 3.0 µl
8 Taq polymerase 1.5U 0.3 µl
Table 4.10.1: Reaction mixture for PCR setup
A total reaction volume of 20µl reaction was set.
The Taq Polymerase was added last to the tube, vortex and spinned briefly,
and then the reaction mixture was carefully layered with 8.0µl of sterile
mineral oil on top of the reaction to prevent evaporation.
These reactions were set up in the sterile hood to avoid any extraneous DNA
contamination from the environment.
cxxxi
The thermal cycler was programmed with times and temperatures for
denaturing, annealing and extending.
Initial denaturation was carried out at 94 C for 13minutes. The cycles were
carried out with the following characteristics.
a. Denaturation 94 C for 30s
b. Annealing 54 C for 30s
c. Extension 72 C for 30s
d. No. of cycles 40
Final extension of 7 min was set for extending temperature. Then at 4oC
until PCR machine was shut off.
When program was completed, the tubes containing the reaction mixture
were taken out and 5 l of gel loading dye was added.
The final PCR product was run on a 1% agarose gel with markers to
determine size of product.
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CHAPTER – 5
RESULTS AND
DISCUSSIONS
cxxxiii
CHAPTER 5
RESULTS AND DISCUSSIONS
5.1. GENE CLONING USING VECTOR NTI SOFTWARE
The software analysis of cloning was successfully carried out using vector
NTI software 11.5 advanced versions. From the software analysis, it was clear that the
pCAMBIA 1301 and the modified gene of interest attached with the NcoI and pmlI
gene sequence at 5’ and 3’end, can be restricted with NcoI and PmlI restriction
enzymes for cloning. Both the plasmid and gene of interest (HCC-scFv) restricted with
the enzyme NcoI and PmlI are ligated to form a clone using this software. The results
of the cloning experiment are shown from Figures 5.1.1 to Figure 5.1.2. The results
from the software analysis (Figure 5.1.7 and Figure 5.1.8) clearly illustrate the
recombinant map and gene sequence which will be obtained after the cloning
experiments.
The Figure 5.1.1 shows the result of anti-HCC scFv antibody gene
sequence downloaded from the Gen bank which was used in vector NTI software for
cloning analysis. The gene map of the downloaded anti-HCC scFv antibody obtained
from the software analysis was show in the Figure 5.1.2. Similarly the results of
modified anti-HCC scFv antibody gene sequence and map are shown in Figure 5.1.3
and Figure 5.1.4 respectively. The results of the gene sequence and the map of the
plasmid vector pCAMBIA 1301 are shown in Figure 5.1.5 and Figure 5.1.6.
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The cloning results obtained from the software analysis using the gene sequence of the
plasmid vector pCAMBIA 1301and the modified anti-HCC scFv antibody are shown
in Figure 5.1.7 and Figure 5.1.8.
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5.1.1. Gene sequence of anti-HCC scFv antibody gene: AY686498
Figure 5.1.1: Analysis of Gene sequence of HCC-scFv: AY686498 downloaded
from the Gen Bank using vector NTI software
5.1.2. Gene Map of anti-HCC scFv antibody gene: AY68649
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Figure 5.1.2: Analysis of Gene Map of HCC-scFv: AY686498 downloaded
from the Gen Bank using vector NTI software
5.1.3. Modified gene sequence of anti-HCC scFv antibody gene: AY686498 with
NcoI and PmlI restriction sites
Figure 5.1.3: Modified Gene sequence of HCC-scFv: AY686498 downloaded from
the Gen Bank using vector NTI software
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5.1.4. Modified gene Map of anti-HCC scFv antibody gene: AY686498 with NcoI
and PmlI restriction sites
Figure 5.1.4: Modified Gene Map of HCC-scFv: AY686498 downloaded from the
Gen Bank using vector NTI software
5.1.5. Gene sequence of pCAMBIA 1301-T-DNA vector
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cxli
cxlii
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Figure 5.1.5: Analysis of Gene sequence of pCAMBIA 1301 downloaded from the
Gen Bank using vector NTI software
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5.1.6. Gene map of pCAMBIA 1301-T-DNA vector
Figure 5.1.6: Analysis of Gene Map of pCAMBIA 1301 downloaded from the Gen
Bank using vector NTI software
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5.1.7. Cloning of anti HCC-scFv into pCAMBIA 1301
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y
Figure 5.1.7: Gene Map: Cloning of pCAMBIA 1301 and HCC-scFv to form
pCAMBIA 1301-HCC-scFv using vector NTI software
5.1.8. Gene sequence of cloned HCC-scFv into pCAMBIA 1301
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cl
cli
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Figure 5.1.8: Gene Sequence: Cloning of pCAMBIA 1301 and HCC-scFv to form
pCAMBIA 1301-HCC-scFvusing vector NTI software
5.2. SYNTHESIS OF HCC-scFv GENE-GENEART
From the results of vector NTI software analysis, the sequence was
submitted to Geneart for gene synthesis. The gene was synthesized, sequenced and
confirmed for the identity of sequence. The percentage of identity of the synthesized
sequence was found to be 100%. The synthesized gene was cloned into pMA-T
containing ampicillin resistance marker and it was received in lyophilized form. These
plasmids were transformed into E. coli DH5α by calcium chloride transformation. The
images from Figure 5.2.1 to Figure 5.2.6 show the results of HCC-scFv gene
synthesis.
The results of the G+C content of the gene sequence analyzed using Gene
Optimizer® was seen in a 40 bp window centered at the indicated nucleotide position.
The average GC content was found to be 54% shown in the Figure 5.2.1, which
indicates the stability of the gene sequence. The Figure 5.2.2 shows the gene sequence
of both the strand used for the synthesis of the gene. The synthesized gene was
sequenced using gene sequencer and the results are shown in Figure 5.2.3. The gene
sequence obtained from the sequencer were compared with the given sequence
(submitted to Geneart) and found to be 100% similar (Figure 5.2.4). The results of
gene sequence and map of the anti-HCC-scFv cloned into pMA-T plasmid vector were
shown in Figure 5.2.5 and Figure 5.2.6.
clvi
5.2.1. Gene sequence analyzed with GeneOptimizer®
The analysis of GC content of the sequence using GeneOptimizer is shown
in the Figure 5.2.1, a 40bp window centered at the indicated nucleotide position. The
average GC content was about 54%.
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Figure 5.2.1: Plot showing G+C content
5.2.2. Gene sequence analysis showing both the strand to be synthesized
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Figure 5.2.2: Gene sequence of both the strands for gene synthesis.
clxi
5.2.3. Analysis of synthesized Gene sequence using Gene sequencer
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Figure 5.2.3: Gene sequenced from the synthesized gene.
5.2.4. Comparitative analysis of the provided gene sequence with the
synthesized gene sequence
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Figure 5.2.4: Sequence identity of the synthesized gene sequence and the given
sequence.
5.2.5. Gene sequence of synthesized gene (HCC-scFv) cloned into pMA-T with
restriction sites.
Figure 5.2.5: Gene sequence of HCC-scFv gene cloned into plasmid pMA-T (pMA-T-
HCC-scFv) with NcoI and PmlI restriction sites.
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5.2.6. Gene construct of HCC-scFv cloned into pMA-T with restriction enzyme
clxix
Figure 5.2.6: Gene Map of pMA-T containing HCC-scFv with various restriction
sites.
5.3. Transformation of plasmid pCAMBIA 1301 and pMA-T into E. coli DH5α
and screening of transformed E. coli DH5α strain
The plasmid pCAMBIA 1301 and pMA-T was successfully transformed
into E. coli (DH5α) by calcium chloride transformation. In case of pCAMBIA 1301
the transformed cells were initially screened on kanamycin antibiotic LB medium. The
transformation efficiency was good which was calculated from the number of colonies
obtained on the plate. The plasmid which was transformed into E. coli (DH5α) carried
kanamycin resistant gene kan and grew on the LB medium containing kanamycin
sulfate (an aminoglycoside antibiotic) shown in the Figure 5.3.1 and Table 5.3.1. The
plates were compared with the controls and the presence of Lac Z gene was also
confirmed using X-gal and IPTG (Figure 5.3.2 and Table 5.3.2). The Lac Z gene
produces β-galactosidase, a stable enzyme which can be easily assayed using the
chromogenic substrate X-gal (5-bromo-4-chloro-indolyl- β-D-galactoside or
galactopyranoside). The X-gal is colourless and when the galactoside moiety is
cleaved off by β-galactosidase, 5-bromo-4-chloro-indolyl is formed and in the
presence of air they form 5-bromo-4-chloro-indigo (blue colour). This colour
formation was observed on the plates indicating that the strains have been transformed
with pCAMBIA 1301 containing lac Z gene.
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Similar to pCAMBIA 1301, the plasmid PMA-T (HCC-scFv) was also
transformed into E. coli (DH5α) by calcium chloride transformation and screened on
amplicillin antibiotic containing LB media. The Figure 5.3.3 and Table 5.3.3 shows
the transformation of pMA-T into E. coli (DH5α). The pMA-T contain ampicillin
resistance gene (amp) that codes for β lactamase, an enzyme that cleaves the β lactam
ring of penicillin and related antibiotics. This enzyme hydrolyzes the amide bond of
the β lactam ring to produce penicilloic acid, which does not show any antibiotic
activity. The E. coli strains transformed with pMA-T produces β lactamase enzyme
and it was secreated into the periplasmic space of the bacterium, where the enzyme
hydrolyzes the β lactam ring and detoxified the ampicillin in the LB media and grew
on the plate.
5.3.1. Transformation of pCAMBIA 1301 and Screening of competent cells
transformed with pCAMBIA 1301 on LB medium (kanamycin)
S.No of Plates
Competent
cells
Concentration
of
antibiotics
E. coli cells
screened for
pCAMBIA
1301
Result
A (positive
control)
200µl
No antibiotics
Untransformed
E. coli DH5α
cells
Lawn of E.
coli DH5α
B (negative
control)
200µl
Kanamycin
(50 µg/ml)
Untransformed
E. coli DH5α
cells
No growth
was
observed
C (experiment 1)
200µl
Kanamycin
(50 µg/ml)
Transformed E.
coli DH5α cells
with pCAMBIA
1301
Transforme
d colonies
grew on the
plate
D (experiment 2)
200µl
Kanamycin
(50 µg/ml)
Transformed E.
coli DH5α cells
with pCAMBIA
1301
Transforme
d colonies
grew on the
plate
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Table 5.3.1: Screening of the transformed E. coli containing pCAMBIA 1301 on
LB medium containing kanamycin.
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Figure 5.3.1: Transformation of pCAMBIA1301 into E. coli DH5α and screening
of the transformed cells on LB medium containing Kanamycin antibiotic.
5.3.2. Screening of transformed E. coli DH5α with pCAMBIA 1301 on LB
medium (kanamycin+X-gal +IPTG)
2
• Transformed cells were screened on antibiotic LB
medium ( - kan, +kan)
A. Untransformed cells-
on LB medium without
Kanamycin
B. Untransformed cells-
on LB medium with
Kanamycin
C and D. Transformed
cells - on LB medium
with Kanamycin
A B
C D
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S. No of Plates
Concentration
of
Antibiotics in
LB medium
E. coli screend for
lac Z gene in
pCAMBIA 1301
Result
A (positive
control)
No antibiotics
Untransformed E.
coli DH5α cells
No bluish green
colonies were
observed
B (negative
control)
Kanamycin
(50 µg/ml)
+
X-gal+IPTG
Untransformed E.
coli DH5α cells
No growth was
observed on the
plate
C (experiment 1)
Kanamycin
(50 µg/ml)
+
X-gal+IPTG
Transformed E. coli
DH5α cells with
pCAMBIA 1301
screened on
kanamycin
Bluish green
colonies were
observed
D (experiment 2)
Kanamycin
(50 µg/ml)
+
X-gal+IPTG
Transformed E. coli
DH5α cells with
pCAMBIA 1301
screened on
kanamycin
Bluish green
colonies were
observed
Table 5.3.2: Screening of the transformed E. coli containing pCAMBIA 1301 on
LB medium containing -kanamycin+X-gal +IPTG.
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Figure 5.3.2: Screening of the E. coli DH5α transformed with pCAMBIA1301 on
LB medium containing Kanamycin, X-gal and IPTG.
X-gal confirmation of transformed E.coli DH5α
• A- non transformed
E.coli DH5α
(white colonies)
• B- no growth
• C- transformed E.coli
DH5α ( blue colonies)
• D- transformed E.coli
DH5α ( blue colonies)
A B
C D
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5.3.3. Transformation of pMA-T:
Plate no Competent
cells
Antibiotics E. coli cells
screened for HCC-
scFv-pMA-T
Result
1 (positive
control)
Competent
cells
200µl
No
antibiotics
No plasmid Lawn of E.
coli DH5α
2 (negative
control)
Competent
cells
200µl
Ampicillin
(100µg/ml)
No plasmid Plate
showed no
growth
3 (experiment 1) Competent
cells
200µl
Ampicillin
(100µg/ml)
Plasmid (HCC-
scFv-pMA-T)
Transforme
d colonies
grew on the
plate
4 (experiment 2) Competent
cells
200µl
Ampicillin
(100µg/ml)
Plasmid (HCC-
scFv-pMA-T)
Transforme
d colonies
grew on the
plate
Table 5.3.3: Screening of the transformed E. coli containing pMA-T-HCC-scFv
on LB medium containing ampicillin.
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Figure 5.3.3: Transformation of pMA-T-HCC-scFv into E. coli DH5α and
screening of the transformed cells on LB medium containing ampicillin antibiotic.
E. coli DH5α transformed with pMA-T-HCC-scFv
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.
5.4. Isolation of plasmid pCAMBIA 1301 and pMA-T from transformed E. coli
DH5α and confirmation by Gel electrophoresis
The plasmid pCAMBIA 1301 and pMA-T was isolated from the
transformed E. coli DH5α and confirmed by gel electrophoresis. The image of the
DNA banding pattern in agrose gel clearly indicates the presence of the plasmid
pCAMBIA 1301 (Figure 5.4.1). The bands formed by pCAMBIA 1301 and pMA-T
on 0.8% agarose gel were shown in the Figure 5.4.2. These bands indicate that the
plasmids have been transformed into the E. coli DH5α cells and the colonies selected
on antibiotic media were found to be transformed with the respective plasmids.
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5.4.1. Gel image of pCAMBIA 1301
ISOLATION OF TRANSFORMED PLASMID PCambia 1301 FROM
E.coli DH5α
• Lane 1-No sample
• Lane 2-5- pCambia 1301
• Lane 6-10-untransformed E.coli DH5α
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Nicked plasmidSupercoiled plasmid
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Figure 5.4.1: Plasmid isolated from transformed E. coli DH5α containing
pCAMBIA 1301.
5.4 2. Gel image of pCAMBIA 1301 and pMA-T (HCC-scFv)
Plasmid isolation from E.coli DH5α transformed with pCambia1301 and pMA-T
• Lane 1-marker
• Lane 2&3 -pCambia 1301 without RNase treatment, Lane 4&5- pCambia 1301 treated with RNase
• Lane 6,7-pMA-T(HCC-scFv) without RNase treatment,Lane 8,9-pMA-T (HCC-scFv) treated with RNase
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
10Kb
8Kb
6Kb
5Kb
4Kb
3Kb
2Kb
1Kb
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Figure 5.4.2: Plasmid isolated from transformed E. coli DH5α (pCAMBIA 1301
and pMA-T-HCC-scFv).
5.5. CLONING OF HCC-scFv FROM pMA-T PLASMID INTO pCAMBIA 1301
5.5.1. Restriction digestion of pCAMBIA 1301 and pMA-T for cloning
The plasmid pCAMBIA1301 and pMA-T (HCC-scFv) was restricted with
NcoI and PmlI restriction enzymes and confirmed on 0.8% agarose gel. From the gel
images it was clear that the restriction digestion of pCAMBIA 1301 by NcoI and PmlI
restriction enzymes formed two bands, shown in the Figure 5.5.1 from lane 2 to 5.
The size of one fragment was about 10kb and another fragment was about 2Kb. The
fragment size of 10Kb was excised from the gel and eluted using gel elution kit for
ligation reaction. This fragment contains the CaMV 35S promoter, hpt marker gene
and Kanamycin antibiotic marker. The 2Kb fragment contain the gus reporter gene,
which was not used in this experiment.
Similarly from the gel images of the restriction digestion of pMA-T (HCC-
scFv) by NcoI and PmlI restriction enzymes, two bands were formed as shown in the
Figure 5.5.1 from lane 6 to 9. One fragment was about 1Kb and another fragment was
about 2.5 Kb. The fragment size of 1Kb was excised from the gel and eluted using gel
clxxxi
elution kit for ligation reaction. This excised fragment contains the gene of interest
(HCC-scFv).
During ligation reaction the 1 Kb fragment of HCC-scFv was ligated with
the 10Kb fragment of pCAMBIA 1301. The 2.5 Kb fragment contains the remaining
plasmid of pMA-T vector. In this experiment pCAMBIA 1301 and pMA-T was
successfully restrict digested with NcoI and PmlI restriction enzymes.
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Figure 5.5.1: Restriction digestion of pCAMBIA 1301 and pMA-T (HCC-scFv)
with NcoI and PmlI restrcyion enzymes
5.5.2. Ligation of HCC-scFv and pCAMBIA 1301
Restriction digestion of pCambia 1301 and pMA-T(HCC-scFv) with NcoI and PmlI
• Lane 1-Marker1Kb
• Lane 2-5- pCambia 1301 restricted with NcoI and PmlI
• Lane 6-9-pMA-T (Hcc-scFv) restricted with NcoI and PmlI
1 2 3 4 5 6 7 8 9 10
10Kb
8Kb5Kb
1Kb
2Kb
3Kb
4Kb
1 2 3 4 5 6 7 8 9 10
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5.5.2.1. Ligation and Transformation of the sample
The fragment size of 9.8020Kb from pCAMBIA 1301 and 0.946Kb of the
Insert DNA was eluted from 0.8% agarose gel by gel elution kit. The eluted linearized
vector DNA- (pCAMBIA 1301-9.802 kb) and the Insert DNA - ( 3:1 molar ratio
excess over vector)-(HCC-scFv-0.946 kb) was efficiently ligated at 22°C (+ 1°C)
using 5X Rapid ligation kit containing T4 DNA Ligase (5U/µl). The ligated samples
were successfully transformed into E. coli DH5α by calcium chloride transformation.
The colonies of E. coli DH5α transformed with ligated vector and DNA insert was
selected on the LB medium containing Kanamycin and the results are shown in the
Figure 5.5.2.1.
5.5.2.2. Confirmation of the ligated clone
The transformed E. coli selected on the kanamycin antibiotic plates were
subcultured by streaking. The plasmid DNA was isolated from the transformed E. coli
cells to confirm the presence of cloned plasmid DNA by alkaline lysis method. The
isolated plasmid was restricted with the same restriction enzymes NcoI and PmlI and
confirmed on 0.8% agarose gel. The agarose gel image in the Figure 5.5.2.2 clearly
shows, the presence of two fragments (9.802 kb and 0.945 kb) in lane 4 confirming
the presence of cloned DNA (pCAMBIA 1301- HCC-scFv).
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Transformation of ligated samples
• Transformed plate 5 (experiment 1) • Transformed colonies were streaked on to the LB
plate containing antibiotic
Figure 5.5.2.1: Transformation of the ligated pCAMBIA 1301 and pMA-T (HCC-
scFv) into E. coli DH5α and screened on LB medium containing Kanamycin.
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Figure 5.5.2.2: Confirmation of the transformed E. coli DH5α containing ligated
pCAMBIA 1301 and pMA-T (HCC-scFv)
Confirmation of the ligated clone
• Lane1: 1 Kb marker
• Lane 2: restricted plasmid (pCambia 1301)- eluted from the gel
• Lane 3: restricted HCC-scFv fragment- eluted from the gel
• Lane 4: restricted pCambia 1301-HCC-scFv(isolated from the transformed ligated sample)
1 2 3 4
10Kb
8Kb
6Kb5Kb
4Kb
3Kb
2Kb
1Kb
clxxxvi
5.6. Transformation of pCAMBIA 1301-scFv from E. coli DH5 α into
Agrobacterium tumefaciens EHA105
The plasmid containing the gene of interest (pCAMBIA 1301-HCC-scFv)
was successfully transformed into Agrobacterium tumefaciens EHA 105. The donor E.
coli DH5α strain harboring Ti plasmid (pCAMBIA1301-HCC-scFv) was transformed
into the recipient strain Agrobacterium tumefaciens EHA 105 with the help of conjugal
helper E. coli HB101 strain harboring pRK2013. The Ti plasmid was mobilized from
donor E. coli DH5α strain into the recipient strain Agrobacterium tumefaciens EHA
105 due to the mobilization function of pRK2013. Agrobacterium tumefaciens EHA
105 harboring the transformed gene was selected on AB minimal medium containing
kanamycin and rifampicin antibiotic. The Ti plasmid contain kanamycin resistant gene
which was used as a marker to screen the transconjugants. Only the transformed
Agrobacterium grew on kanamycin antibiotic selection medium. In this experiment the
plasmid pCAMBIA1301-HCC-scFv from the E. coli DH5α was transformed into
Agrobacterium tumefaciens EHA 105 by triparental mating. The results of triparental
mating are shown in the Figure 5.6.1 and Figure 5.6.2. These transformed
Agrobacterium tumefaciens EHA 105 were used for Agrobacterium-mediated
transformation, to transfer gene into IR64 rice variety.
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Figure 5.6.1: Transformation of pCAMBIA 1301-HCC-scFv into A.tumefaciens EHA
105 by triparental mating and screening on AB minimal medium containing
Kanamycin antibiotic
TRANSFORMATION OF pCAMBIA 1301-HCC-scFv INTO AGROBACTERIUM
EHA 105 BY TRIPARENTAL MATING
• Agrobacterium EHA 105 selected on
rifampicin medium
• Transformed Agrobacterium EHA 105
selected on AB minimal medium with
Rifampicin and kanamycin antibiotic
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Transformation of pCambia 1301-HCC-scFv into Agrobacterium
tumefaciens EHA 105 by Triparental mating
E.coli DH5α-
(pCambia 1301-
HCC-scFv)
E.coli HB101
(pRK2013)
Agrobacterium tumefaciens EHA 105
TRIPARENTAL
MATING
Transformed A.tumefaciens EHA 105
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Figure 5.6.2: Transformation of pCAMBIA 1301-HCC-scFv into A.tumefaciens EHA
105 by triparental mating.
5.7. Callus Induction of IR64 rice variety for transformation
White soft embryogenic callus developed on the surface of the rice scutellar
region after 2 to 3 weeks. The high concentration of 2, 4-D (2mg/L) induced the
formation of callus (undifferentiated mass of cells) and inhibited the formation of
roots. The seeds obtained from ICAR and callus obtained after 2 to 3 weeks are shown
in the Figure 5.7.1.
The phenomenon of the reversion of mature cells to the meristematic state
leading to the formation of callus is called dedifferentiation. These dedifferentiated
cells have the capacity to form a whole plant, a phenomenon described as
redifferentiation. These two phenomenons of dedifferentiation and redifferentiation are
described as cellular totipotency, which is seen only in plants and not in animal cells.
These dedifferentiated cells (callus) can be transformed with the gene of interest by
Agrobactetium – mediated transformation and they can be regenerated.
In this experiment callus has been successfully induced from IR64 rice
variety. The callus induced from IR64 rice variety can be used for Agrobacterium
mediated transformation to transfer the HCC-scFv gene.
cxc
cxci
Figure 5.7.1: Callus induction of IR4 using 2, 4 D-MS medium
5.8. Transformation of pCAMBIA 1301-HCC-scFv from Agrobacterium
tumefaciens EHA 105 into callus by Agrobacterium- mediated transformation
Callus induction of Orzae sativa –Indica rice variety- IR 64
callus
IR 64 obtained
from ICAR,
Cuttack,India
Callus induction on 2,4 D
MS medium
Callus obtained on 2,4 D MS
medium
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The callus which was infected with Agrobacterium was selected on
first selection medium using cefotaxime. The surviving calli were further seleceted on
second selection medium containing cefotaxime and hygromycin B. The image of
third round of selection using cefotaxime and hygromycin B is shown in the Figure
5.8.1. The healthy surviving embryogenic calli was transferred into MSKN
regeneration medium. The image of regeneration has been shown in the Figure 5.8.1.
Cefotaxime was used to prevent contamination and kill bacteria. The callus
which was transformed with the Agrobacterium gene carrying pCAMBIA1301-HCC-
scFV containing the hpt gene which codes for hygromycin phosphotransferase
breakdown the aminocyclitol antibiotic hygromycin B in the substrate and grow on the
media. In the absence of transformation of the gene, the callus cannot break the
aminocyclitol antibiotic hygromycin B in the substrate due to lack of hpt gene and
thus, the callus does not survive on the media.
In this experiment, the gene of interest (pCAMBIA1301-HCC-scFV) was
successfully transformed from Agrobacterium tumefaciens EHA 105 into callus of IR
64 and it was successfully regenerated.
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Figure 5.8.1: Transformation of pCAMBIA 1301-HCC-scFv from A.tumefaciens
EHA 105 into IR64 and selection of the transformed callus on Hygromycin.
5.9. Extraction of total genomic DNA from plant tissues for Molecular Analysis
Transformation of pCambia 1301-HCC-scFv from Agrobaerium tumefaciens
EHA 105 into callus of IR 64 and regeneration
• Third round of calli selection on
cefotaximine and hygromycin
• Regeneration of callus
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The total genomic DNA from transformed and untransformed leaf samples
were extracted using CTAB method. A white precipitate was obtained which was
dissolved in sterile water and stored at -20°C. The amount of DNA obtained was
measured using Biophotometer. The organic extraction is a conventional technique
and the solvents are used to extract the contaminants from the cell lysate. The cells
were lysed by CTAB-detergent and then mixed with phenol, chloroform and isoamyl
alcohol. The concentration of salt and the pH used during the extraction must be
correct to ensure that contaminats are separated into the organic aqueous and the DNA
remains in the aqueous phase. DNA is precipitated and recovered by alcohol
precipitation. This technique is time consuming and the isolated DNA may contain
residual phenol or chloroform which might inhibit the downstream processing of the
PCR. Therefore purification of the DNA is required for further downstream
processing.
The isolated DNA was purified using the PCR clean up kit and the amount
of DNA obtained was measured using Biophotometer. Purification of the sample was
carried out to remove any residual components left behind after DNA isolation. The
purified samples were used for the detection and confirmation of CaMV35S promoter
gene sequence using PCR.
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5.10. Molecular analysis of Putative transformed plants by Polymerase Chain
Reaction (PCR)
The sequence of the forward primer (FP-19bp long- CaMV35S) and the
reverse primer (RP-20bp long- CaMV35S) are specific for CaMV35S promoter gene
sequence, present upstream of the cloned pCAMBIA 1301-HCC-scFv gene. These
primers would bind specifically to the CaMV35S promoter gene sequence region of
the template DNA based on the homology and would help in the amplification of the
amplicon (195bp). As the result of amplification a minute concentration of template
DNA would get amplified to 1000 folds at the end of 30 cycles.
From the agarose gel image A (Figure 5.10.1), negative control 1 did not
show any band indicating the purity of water and the reaction mixture. The negative
control 2 did not show any band indicating the absence of the CaMV35S gene in
untransformed control plant. The positive control in lane 4 and 5 showed a thick band
indicating the presence of CaMV35S promoter gene and proves that the reaction
conditions were perfect. The lanes from 6 to 10 showed thick band indicating the
presence of CaMV35S in the transformed and regenerated plant. All the samples
showed amplification of CaMV35S.
Similar results were seen in the Agarose gel image B (Figure 5.10.1), Lane
10 showed the marker band, Lane 6 and 7 showed the amplification of the positive
control, Lane 8 and 9 did not show any amplification indicating untransformed
regenerated plant and Lane 1 to 5 showed the amplification of CaMV35S promoter
gene indicating that the plant has been transformed with T-DNA of pCAMBIA 1301
containing the gene of interest (HCC-scFv).
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Figure 5.10.1: Molecular analysis of the transformed plant (CaMV35S) by
polymerase chain reaction
PCR confirmation of CaMV35 gene in regenerated leaves
• Lane 1- marker (100bp)
• Lane 2-Negative control 1
• Lane 3-Negative control 2
• Lane 4&5-Positive control
• Lane 6-10- Samples-amplified CaMV 35S
• Lane 1-5- transformed callus regenerated-amplified CaMV 35S
• Lane 6,7-Positive control
• Lane 8,9- Negative control 3-untransformed callus regenerated
• Lane 10-marker(100bp)
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
195bp200 bp
100 bp
1000 bp
900 bp
800 bp
700 bp
600 bp
300 bp
400 bp500 bp
1000 bp
100 bp
A B
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.
CHAPTER – 6
SUMMARY
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CHAPTER 6
SUMMARY
The cloning of plasmid pCAMBIA 1301 and HCC-scFv (gene of interest) was
initially carried out using vector NTI software. The 5’ end and 3’ end of the
gene of interest (HCC-scFv) was modified by attaching with the gene sequence
of signal peptide (Rice α amylase) at 5’ end, KDEL retension signal peptide at
3’ end and the two adaptors NcoI (5’end) and pmlI (3’ end) were attached to the
gene to facilitate cloning. Cloning was initially carried out in vector NTI
software to confirm if the gene sequence could be inserted into the chosen T-
DNA plasmid – pCAMBIA 1301.
The gene of interest HCC-scFv from gene bank accession no: AY686498 had to
be modified to clone the gene into plant expression system based on the results
of the vector NTI software. The 5’ end and 3’ end were modified by adding
gene sequence of rice α amylase signal peptide at 5’end at 1-60 bp positions in
AY686498 and endoplasmic reticulum retention signal peptide KDEL at 3’end
to retain the protein molecule inside the endoplasmic reticulum and to avoid
protein degradation in the cytoplasm by proteolytic enzymes. To overcome the
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limitation of plant N-glycosylation, the proteins were retained in the
endoplasmic reticulum.
The ends were further modified by attaching the gene sequence of NcoI
adaptors and Pml adaptors to obtain restriction site present in the plasmid
pCAMBIA1301. In the modified AY686498 gene sequence the 24th
code c
(cytosine) i.e. cca coding for theronine was replaced by cga which also codes
for theronine. The reason for changing the coding sequence: ccatgg was
because that the gene sequence will be restricted by NcoI, when changed to
cgatgg the restriction site is altered but the amino acid sequence will remain the
same. This helped to clone the gene into pCAMBIA 1301 at NcoI and PmlI
restriction sites were the entire cDNA strand will remain unrestricted, when
restriced with NcoI and pmlI restriction enzymes.
The gene was cloned into pCAMBIA 1301 and transformed from E. coli DH5α
into Agrobacterium tumefaciens EHA 105 which was further co-cultivated with
the callus induced by 2, 4-D. The transformed regenerated plant was screened
for the presence of CaMV35S promoter. The amplification of CaMV35S from
the transformed plant indicates the presence of the HCC-scFv which was cloned
downstream of CaMV35S promoter.
cc
CHAPTER – 7
cci
CONCLUSION AND
SCOPE OF FUTURE
WORK
CHAPTER 7
CONCLUSION AND SCOPE OF FUTURE WORK
7.1. CONCLUSION
ccii
The plasmid pCAMBIA 1301 and HCC-scfv (gene of interest) was successfully
cloned using vector NTI software after modifying the 5’ and 3’ ends of HCC-
scFv gene sequence.
The HCC-scFv gene sequence analysed for cloning, in vector NTI software was
successfully synthesized with the modified ends, to clone and express the gene
in the plant expression system.
The plasmid pCAMBIA 1301 (vector) and pMA-T (containing HCC-scFv)
obtained were successfully transformed into E. coli (DH5α) by calcium chloride
transformation. The transformed strains were confirmed by isolating the
plasmid and running them on the agarose gel.
The pCAMBIA 1301 (T-DNA vector) and pMA-T (containing the gene of
interest –HCC-scFv) were restricted with NcoI and PmlI restriction enzymes
and confirmed on 0.8% Agarose.
The band of particular size of the plasmid and the insert was successfully
eluted, ligated, transformed and confirmed on the agarose gel. From this
experiment it was confirmed that the gene of interest HCC-scFv was cloned
into the plasmid pCAMBIA 1301 and the cloned plasmid was denoted as
pCAMBIA 1301-HCC-scFv.
The recombinant Ti plasmid containing the gene of interest (pCAMBIA 1301-
HCC-scFv). was mobilized from E. coli DH5α into Agrobacterium tumefaciens
EHA 105
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Callus has been successfully induced using 2, 4-D on MS medium containing
organic salts, inorganic salts, vitamins and carbon sources.
The Agrobacterium tumefaciens EHA 105 containing pCAMBIA1301-HCC-
scFv was successfully transformed into IR 64 callus and it was successfully
regenerated.
The DNA was isolated from the plant tissue by CTAB method and it was
further purified by using PCR clean up kit.
From the PCR technique it is clear that the T-DNA of pCAMBIA 1301
containing CaMV35S has been transformed into plant along with the gene of
interest (HCC-scFv). As HCC-scFv gene was cloned after CaMV35S promoter,
the amplification of CaMV35S indicates the presence of the HCC-scFv.
7.2. APPLICATION
The HCC-scFv transformed into IR64 can be used for the diagnosis and
immunotherapy of Heptocellular carcinoma (HCC).
The gene of HCC-scFv fragment cloned into IR64 can specifically bind to the
receptors of the Heptocellular carcinoma (HCC) cells.
The protein expressed from the cloned IR64 containing HCC-scFv gene
fragment can be isolated from the transformed plants which can be fused with
pro-drug, anti-cancer agents or radio nucleides. This fused HCC-scFv can
specifically bind to hepatocellular carcinoma receptors and release the
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particular drug specifically to the Heptocellular carcinoma (HCC) cells and kills
them.
The HCC-scFv fragment can be isolated from the transformed IR64 rice variety
which can also be used for the diagnosis of Heptocellular carcinoma (HCC) in
the earlier stages of cancer.
The method used in this research work can be used to produce various other
scFv fragments to target against other cancer receptors for diagnostic purposes
and immunotheraphy in IR64 rice variety.
7.3. SCOPE FOR FUTURE WORK
The HCC-scFv protein has to be extracted from the transformed IR64 rice
variety by immobilized metal affinity chromatoghraphy using IMAC
purification kit (5X his tag (fused with the gene) or 6X his tag (present on the
plasmid).
The presence of the protein has to be confirmed by western blotting.
The isolated protein (human monoclonal HCC-scFv) confirmed by western
blotting, can be fused with the drugs targeted specifically to HCC.
The drug fused protein has to be checked for its specificity on HepG2 cell line.
Toxicity assessment of the human monoclonal HCC-scFv fused with the drug
has to be carried out for its application.
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APPENDICES
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APPENDICES
Appendix 1
Equation 4.5.2.1
Equation 4.7.1
Equation 4.8.1
Equation 4.10.1
Tm = 4 C (No. of G+C) + 2 C (No. of A+T)
ng of insert = ng of vector X Size of insert X insert ratio
--------------------------------
Size of vector X Vector ratio
Callus Induction frequency % = Total number of seeds that produced callus x 100
Total number of seeds plated
CRF (%) = Total number of callus that produced plants
---------------------------------------------------- X 100
Total number of callus plated
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Appendix 2
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Appendix 3
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LIST OF
PUBLICATIONS
LIST OF PUBLICATIONS
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(Peer-Reviewed Publications- International journals)
Aadarsh Prasanna, Deepa.V, Balakrishna Murthy.P, M.Deecaraman, Sridhar.R
and Dhandapani P. Insoluble phosphate solubilization by Bacterial strains
isolated from Rice rhizosphere soils from Southern India. International journal of
soil science., 2011, 6(2), 134-141. (Impact factor 0.4)
Aadarsh Prasanna, P. Balakrishna Murthy, M. Deecaraman and R. Sridhar.
Optimization of Callus Induction and Regeneration in Swarna masoori rice
cultivar. Research Journal of Agriculture and Biological Sciences, 6(6): 917-922,
2010. (Impact factor 0.3)
Deepa.V, Aadarsh Prasanna, Balakrishna Murthy.P, M.Deecaraman, Sridhar.R
Efficient Phosphate solubilization by fungal strains isolated from Rice-
rhizosphere soils for the phosphorus release. Research journal of Agriculture
and Biological Science, 6(4): 487-492, 2010. (Impact factor 0.3)
T.N. Sathya, Aadarsh Prasanna, Deepa.V, Balakrishna Murthy.P. Moringa
oleifera lam. Leaves prevent Cyclophosphamide-induced micronucleus and DNA
damage in mice. International Journal of phytomedicine, 2 (2010) 147-154. (
Impact factor 0.1)
