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Robert Entus Motif Based Network Analysis and Design
Synthetic Biology Network Analysis and Design
Robert Entus, Ph.D.
Robert Entus Motif Based Network Analysis and Design
“Synthesis drives discovery and paradigm change in ways not possible through
analysis”
-Steve Brenner
Robert Entus Motif Based Network Analysis and Design
What is Synthetic Biology?
There are two fundamental research areas that fall under the term:
• Retooling biological interactions on the molecular scale by developing synthetic analogs to biological molecules, e.g. novel bases that allows Watson-Crick base pairing in DNA to have more than four bases.
• Using existing biological components to the increase the understanding of cellular biology and the requirements for adding functionality. Combines knowledge from many disciplines, such as molecular biology, computational sciences, and engineering.
Robert Entus Motif Based Network Analysis and Design
What is Synthetic Biology?
There are two fundamental research areas that fall under the term:
• Retooling biological interactions on the molecular scale by developing synthetic analogs to biological molecules, e.g. novel bases that allows Watson-Crick base pairing in DNA to have more than four bases.
• Using existing biological components to the increase the understanding of cellular biology and the requirements for adding functionality. Combines knowledge from many disciplines, such as molecular biology, computational sciences, and engineering.
Robert Entus Motif Based Network Analysis and Design
Advantages of Synthetic Biology?
• What can SB do for us?• Small gene networks are
introduced into hosts, such as E. coli to study existing networks and motifs by altering individual elements.
• develop novel and increasingly complex gene networks in single cell and multi cellular systems
• development of sophisticated behaviors such as bistable switches, oscillators, biosensors, drug synthesis, and programmable pattern formation.
Robert Entus Motif Based Network Analysis and Design
Advantages of Synthetic Biology?
• What can SB do for us?• Small gene networks are
introduced into hosts, such as E. coli to study existing networks and motifs by altering individual elements.
• develop novel and increasingly complex gene networks in single cell and multi cellular systems
• development of sophisticated behaviors such as bistable switches, oscillators, biosensors, drug synthesis, and programmable pattern formation.
Robert Entus Motif Based Network Analysis and Design
Advantages of Synthetic Biology?
• What can SB do for us?• Small gene networks are
introduced into hosts, such as E. coli to study existing networks and motifs by altering individual elements.
• develop novel and increasingly complex gene networks in single cell and multi cellular systems
• development of sophisticated behaviors such as bistable switches, oscillators, biosensors, drug synthesis, and programmable pattern formation.
Robert Entus Motif Based Network Analysis and Design
Advantages of Synthetic Biology?
• What can SB do for us?• Small gene networks are
introduced into hosts, such as E. coli to study existing networks and motifs by altering individual elements.
• develop novel and increasingly complex gene networks in single cell and multi cellular systems
• development of sophisticated behaviors such as bistable switches, oscillators, biosensors, drug synthesis, and programmable pattern formation.
Robert Entus Motif Based Network Analysis and Design
Advantages of Synthetic Biology?
• What can SB do for us?• Small gene networks are
introduced into hosts, such as E. coli to study existing networks and motifs by altering individual elements.
• develop novel and increasingly complex gene networks in single cell and multi cellular systems
• development of sophisticated behaviors such as bistable switches, oscillators, biosensors, drug synthesis, and programmable pattern formation.
Robert Entus Motif Based Network Analysis and Design
Network SimplificationWithout simplification understanding network interactions becomes
very difficult
Example: Given b = 3.6 what does a =?
a4 + 4a3b + 6a2b2 + 4ab3 + b4 = 1296
Robert Entus Motif Based Network Analysis and Design
Network SimplificationWithout simplification understanding network interactions becomes
very difficult
Example: Given b = 3.6 what does a =?
a4 + 4a3b + 6a2b2 + 4ab3 + b4 = 1296
With simplification the solution becomes trivial
Solution: (a + b)4 = 64
a + 3.6 = 6 a = 2.4
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 metJGFP
Arabinose
GFP
MetJ
Network For Feedback Amplifier
Robert Entus Motif Based Network Analysis and Design
How To Create Synthetic Network
• Define experimental conditions. What host will you be using, E.coli, yeast, or mammalian, how will you measure the output, etc.
• Develop a working model of the network that contains necessary and appropriate components, specifically a measurable input and output. – No matter how useful a protein appears to be, in your design, if
its production kills the host, it is not a good choice.
• Construct the network with experimental conditions in mind. Will you want to switch hosts later on down the line?
Robert Entus Motif Based Network Analysis and Design
How To Create Synthetic Network
• Define experimental conditions. What host will you be using, E.coli, yeast, or mammalian, how will you measure the output, etc.
• Develop a working model of the network that contains necessary and appropriate components, specifically a measurable input and output. – No matter how useful a protein appears to be, in your design, if
its production kills the host, it is not a good choice.
• Construct the network with experimental conditions in mind. Will you want to switch hosts later on down the line?
Robert Entus Motif Based Network Analysis and Design
How To Create Synthetic Network
• Define experimental conditions. What host will you be using, E.coli, yeast, or mammalian, how will you measure the output, etc.
• Develop a working model of the network that contains necessary and appropriate components, specifically a measurable input and output. – No matter how useful a protein appears to be, in your design, if
its production kills the host, it is not a good choice.
• Construct the network with experimental conditions in mind. Will you want to switch hosts later on down the line?
Robert Entus Motif Based Network Analysis and Design
General Experimental Setup
• Synthetic Networks were designed and assembled on plasmids capable of expression in E.coli.
• Cells expressing the network of interest were grown overnight, diluted 1:300 into LB with the appropriate supplements and grown to an OD600=0.6.
Robert Entus Motif Based Network Analysis and Design
General Experimental Setup
• Synthetic Networks were designed and assembled on plasmids capable of expression in E.coli.
• Cells expressing the network of interest were grown overnight, diluted 1:300 into LB with the appropriate supplements and grown to an OD600=0.6.
Robert Entus Motif Based Network Analysis and Design
General Experimental Setup
• The cells were washed and resuspended in M9 Medium and transferred to a 96 well plate and covered with mineral oil.
• Fluorescent (ex. 395, em. 515), and OD600 measurements were taken every 8 minutes with orbital shaking between measurements.
Clear Bottom 96 Well Plate Victor 3 Plate Reader
Robert Entus Motif Based Network Analysis and Design
General Experimental Setup
• The cells were washed and resuspended in M9 Medium and transferred to a 96 well plate and covered with mineral oil.
• Fluorescent (ex. 395, em. 515), and OD600 measurements were taken every 8 minutes with orbital shaking between measurements.
Clear Bottom 96 Well Plate Victor 3 Plate Reader
Robert Entus Motif Based Network Analysis and Design
PNAS (2003) 100:7702
Plate Based System
• Measure samples through out entire experiment. No need to use additional protocols to determine network state.
• Robust experimental conditions possible in a single run.
• High throughput experimentation possible
• Small footprint• Low experimental costs
Robert Entus Motif Based Network Analysis and Design
PNAS (2003) 100:7702
Plate Based System
• Measure samples through out entire experiment. No need to use additional protocols to determine network state.
• Robust experimental conditions possible in a single run.
• High throughput experimentation possible
• Small footprint• Low experimental costs
Robert Entus Motif Based Network Analysis and Design
PNAS (2003) 100:7702
Plate Based System
• Measure samples through out entire experiment. No need to use additional protocols to determine network state.
• Robust experimental conditions possible in a single run.
• High throughput experimentation possible
• Small footprint• Low experimental costs
Robert Entus Motif Based Network Analysis and Design
PNAS (2003) 100:7702
Plate Based System
• Measure samples through out entire experiment. No need to use additional protocols to determine network state.
• Robust experimental conditions possible in a single run.
• High throughput experimentation possible
• Small footprint• Low experimental costs
Robert Entus Motif Based Network Analysis and Design
PNAS (2003) 100:7702
Plate Based System
• Measure samples through out entire experiment. No need to use additional protocols to determine network state.
• Robust experimental conditions possible in a single run.
• High throughput experimentation possible
• Small footprint• Low experimental costs
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 metJGFP
Arabinose
GFP
MetJ
Working Model
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
AraC binds araI and O2, bending the DNA so that the two DNA bound AraC contact each other
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
Arabinose
Arabinose causes a conformation change allowing AraC to bind araI and O1 allowing the transcription
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 GFP
Arabinose
the operator sites OR1 and OR2 cooperatively bind cI, which in turn acts as a transcriptional activator by recruiting RNA polymerase.
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 GFP
Arabinose
GFP
The operator sites OR1 and OR2 cooperatively bind cI, which in turn acts as a transcriptional activator by recruiting RNA polymerase.
GFP is produced as the measurable output.
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 GFP
Arabinose
GFP
AGgatTtT AGcCGTCc AGAtGTtT AcACaTCc
AGgatTtT AGcCGTCc AGAtGTtT AcACaTCc 50% 75% 75% 63%
Tandem “met box” sequences provide a repressor binding site
metJ
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 metJGFP
Arabinose
GFP
MetJ
AGgatTtT AGcCGTCc AGAtGTtT AcACaTCc
metJ is transcribed from the same mRNA as GFP.
Robert Entus Motif Based Network Analysis and Design
MetJ provides negative feedback in the system by preventing cI production.
AraC
O1O2 araI cI
cI
OR1 OR2 metJGFP
Arabinose
GFP
MetJ
Robert Entus Motif Based Network Analysis and Design
MetJ induced negative feedback can be modulated in two different areas.
AraC
O1O2 araI cI
cI
OR1 OR2 metJGFP
Arabinose
GFP
AGgatTtT AGcCGTCc AGAtGTtT AcACaTCc
The Ribosome Binding Site controlling MetJ translation.
Each 8 base sequence can be altered independently.
Robert Entus Motif Based Network Analysis and Design
Network Construction
• Building synthetic networks requires an understanding of fundamental molecular biology procedures:– Vector selection– Restriction enzymes to produce compatible ends– Gene cloning (Polymerase Chain Reaction)– Ligation reactions
Robert Entus Motif Based Network Analysis and Design
Vector Selection
The completed network will look like this.
The network will be built on a single circular plasmid that contains all of the network components
Not this.
Robert Entus Motif Based Network Analysis and Design
Restriction Enzymes• Restriction enzymes cut the DNA
at specific palindromic sites that range from 4 to 8 bases.
• Many restriction enzymes leave “sticky” overhangs that allow directed cloning.
• Several restriction enzymes can be used in the same reaction, as long as their buffer requirements are compatible.
• The ability to cut a restriction site is dependent on many factors: the methylation state, secondary structures, and proximity to the end. The activity of most enzymes decreases dramatically when you get closer than six bases from the end.
Crystal Structure of Eco RI bound to DNA substrate.
Recognition Sequence.
Robert Entus Motif Based Network Analysis and Design
Restriction Enzymes• Restriction enzymes cut the DNA
at specific palindromic sites that range from 4 to 8 bases.
• Many restriction enzymes leave “sticky” overhangs that allow directed cloning.
• Several restriction enzymes can be used in the same reaction, as long as their buffer requirements are compatible.
• The ability to cut a restriction site is dependent on many factors: the methylation state, secondary structures, and proximity to the end. The activity of most enzymes decreases dramatically when you get closer than six bases from the end.
Crystal Structure of Eco RI bound to DNA substrate.
Recognition Sequence.
Robert Entus Motif Based Network Analysis and Design
Restriction Enzymes• Restriction enzymes cut the DNA
at specific palindromic sites that range from 4 to 8 bases.
• Many restriction enzymes leave “sticky” overhangs that allow directed cloning.
• Several restriction enzymes can be used in the same reaction, as long as their buffer requirements are compatible.
• The ability to cut a restriction site is dependent on many factors: the methylation state, secondary structures, and proximity to the end. The activity of most enzymes decreases dramatically when you get closer than six bases from the end.
Crystal Structure of Eco RI bound to DNA substrate.
Recognition Sequence.
Robert Entus Motif Based Network Analysis and Design
Restriction Enzymes• Restriction enzymes cut the DNA
at specific palindromic sites that range from 4 to 8 bases.
• Many restriction enzymes leave “sticky” overhangs that allow directed cloning.
• Several restriction enzymes can be used in the same reaction, as long as their buffer requirements are compatible.
• The ability to cut a restriction site is dependent on many factors: the methylation state, secondary structures, and proximity to the end. The activity of most enzymes decreases dramatically when you get closer than six bases from the end.
Crystal Structure of Eco RI bound to DNA substrate.
Recognition Sequence.
Robert Entus Motif Based Network Analysis and Design
Restriction Enzymes
Nde I digestion
-CATATG--GTATAC-
-CA TATG--GTAT AC-
Sal I digestion
- GTCGAC -- GTCGAC -
- G TCGAC -- GTCGA C -
Two common restriction sites that allows directed cloning. Although many enzymes are available, Nde I is often used (even though it has minor problems) due to fact that the site incorporates a terminal ATG that provides the start codon in protein synthesis.
Robert Entus Motif Based Network Analysis and Design
Common Restriction EnzymesEnzyme Source Recognition Sequence Cut
EcoRI Escherichia coli 5'GAATTC3'CTTAAG 5'---G AATTC---3
BamHI Bacillus amyloliquefaciens 5'GGATCC3'CCTAGG 5'---G GATCC---3'
HindIII Haemophilus influenzae 5'AAGCTT3'TTCGAA 5'---A AGCTT---3'
TaqI Thermus aquaticus 5'TCGA3'AGCT 5'---T CGA---3
NotI Nocardia otitidis 5'GCGGCCGC3'CGCCGGCG 5'---GC GGCCGC---3’
Sau3A Staphylococcus aureus 5'GATC3'CTAG 5'--- GATC---3'
PovII* Proteus vulgaris 5'CAGCTG3'GTCGAC 5'---CAG CTG---3'
SmaI* Serratia marcescens 5'CCCGGG3'GGGCCC 5'---CCC GGG---3'
HaeIII* Haemophilus egytius 5'GGCC3'CCGG 5'---GG CC---3
AluI* Arthrobacter luteus 5'AGCT3'TCGA 5'---AG CT---3
EcoRV* Escherichia coli 5'GATATC3'CTATAG 5'---GAT ATC---3'
KpnI[2] Klebsiella pneumonia 5'GGTACC3'CCATGG 5'---GGTAC C---3'
PstI[2] Providencia stuartii 5'CTGCAG3'GACGTC 5'---CTGCA G---3'
SacI[2] Streptomyces achromogenes 5'GAGCTC3'CTCGAG 5'---GAGCT C---3'
SalI[2] Streptomyces albue 5'GTCGAC3'CAGCTG 5'---G TCGAC---3'
Robert Entus Motif Based Network Analysis and Design
Ligation Reaction
• Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA.
• Ligases can join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.
Robert Entus Motif Based Network Analysis and Design
lac promoter
NdeI restricition site (CATATG)
Sal I restriction site (GTCGAC)
pBR322 plasmid with lac promoter
Double digest withNde I and Sal I
lac promoter
NdeI overhangSal I overhang
Nde I digestion
-CATATG--GTATAC-
-CA TATG--GTAT AC-
Sal I digestion
- GTCGAC -- GTCGAC -
- G TCGAC -- GTCGA C -
Robert Entus Motif Based Network Analysis and Design
GFP gene
NdeI restricition site (CATATG)
Sal I restriction site (GTCGAC)
6 bp extension
6 bp extension
PCR Amplification
GFP gene with new restriction sites
Double digest withNde I and Sal I
Robert Entus Motif Based Network Analysis and Design
lac promoter GFP
Ligation reaction
repeat process for additional genes
Robert Entus Motif Based Network Analysis and Design
Now What?
• Now that we have our network and experimental setup complete we can sit back and let the data come rolling in.
• As with all experiments, conditions will need to tailored to fit the needs, the network will need adjustments, and data stored/evaluated
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 metJGFP
Arabinose
GFP
MetJ
Network For Signal Linearization
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI GFP
Arabinose
GFP
Network For Signal Linearization
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
0.0001 0.001 0.01 0.1 1 10
Arabinose (%)
Rel
ativ
e F
luo
resc
ence
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI
metJ
GFP
Arabinose
GFP
MetJ
Network For Signal Linearization
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
0.0001 0.001 0.01 0.1 1 10
Arabinose (%)
Rel
ativ
e F
luo
resc
ence
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI metJGFP
Arabinose
GFP
MetJ
Network For Signal Linearization
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
0.0001 0.001 0.01 0.1 1 10
Arabinose (%)
Rel
ativ
e F
luo
resc
ence
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 GFP
Arabinose
GFP
Network For Signal Linearization
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
0.0001 0.001 0.01 0.1 1 10
Arabinose (%)
Rel
ativ
e F
luo
resc
ence
Robert Entus Motif Based Network Analysis and Design
AraC
O1O2 araI cI
cI
OR1 OR2 metJGFP
Arabinose
GFP
MetJ
Network For Signal Linearization
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10
p1
p3
Feed Forward Loop Model
Increasing Repression
Robert Entus Motif Based Network Analysis and Design
RNAP
Unregulated Networks
• T7 RNA polymerase (RNAP) is under the control of the lac promoter.
• Increasing Isopropyl-β-D-thiogalactopyranoside (IPTG) results in increasing amounts of RNAP being produced.
• A T7 binding site is located ~70 bases upstream of the Green Fluorescent protein (GFP) start sequence.
• GFP (ex. 395 nm, em. 515 nm) is the measured output
Robert Entus Motif Based Network Analysis and Design
RNAP
IPTG
Unregulated Networks
• T7 RNA polymerase (RNAP) is under the control of the lac promoter.
• Increasing Isopropyl-β-D-thiogalactopyranoside (IPTG) results in increasing amounts of RNAP being produced.
• A T7 binding site is located ~70 bases upstream of the Green Fluorescent protein (GFP) start sequence.
• GFP (ex. 395 nm, em. 515 nm) is the measured output
Robert Entus Motif Based Network Analysis and Design
TAATACGACTCACTATA
RNAP
IPTG
T7 GFP
Unregulated Networks
• T7 RNA polymerase (RNAP) is under the control of the lac promoter.
• Increasing Isopropyl-β-D-thiogalactopyranoside (IPTG) results in increasing amounts of RNAP being produced.
• A T7 binding site is located ~70 bases upstream of the Green Fluorescent protein (GFP) start sequence.
• GFP (ex. 395 nm, em. 515 nm) is the measured output
Robert Entus Motif Based Network Analysis and Design
TAATACGACTCACTATA
RNAP
IPTG
GFP
T7 GFP
Unregulated Networks
• T7 RNA polymerase (RNAP) is under the control of the lac promoter.
• Increasing Isopropyl-β-D-thiogalactopyranoside (IPTG) results in increasing amounts of RNAP being produced.
• A T7 binding site is located ~70 bases upstream of the Green Fluorescent protein (GFP) start sequence.
• GFP (ex. 395 nm, em. 515 nm) is the measured output
Robert Entus Motif Based Network Analysis and Design
RNAP
IPTG
T7 GFP
Unregulated Networks
• The metR promoter has been inserted between the T7 RNAP binding site and the GFP start site.
• The met operator in E.coli consists of tandem repeats of eight base pair sequences, homologous to a palindromic consensus AGACGTCT, known as “met boxes” to downregulate transcription.
• There are 4 met boxes in the metR promoter of E.coli. The sequences correspond to a 50%, 75%, 75%, and 63% identity to the consensus sequence.
Robert Entus Motif Based Network Analysis and Design
RNAP
IPTG
T7 GFPmetRpro
Unregulated Networks
• The metR promoter has been inserted between the T7 RNAP binding site and the GFP start site.
• The met operator in E.coli consists of tandem repeats of eight base pair sequences, homologous to a palindromic consensus AGACGTCT, known as “met boxes” to downregulate transcription.
• There are 4 met boxes in the metR promoter of E.coli. The sequences correspond to a 50%, 75%, 75%, and 63% identity to the consensus sequence.
Robert Entus Motif Based Network Analysis and Design
RNAP
IPTG
T7 GFPmetRpro
Unregulated Networks
• The metR promoter has been inserted between the T7 RNAP binding site and the GFP start site.
• The met operator in E.coli consists of tandem repeats of eight base pair sequences, homologous to a palindromic consensus AGACGTCT, known as “met boxes” to downregulate transcription.
• There are 4 met boxes in the metR promoter of E.coli. The sequences correspond to a 50%, 75%, 75%, and 63% identity to the consensus sequence.
AGgatTtT AGcCGTCc AGAtGTtT AcACaTCc
Robert Entus Motif Based Network Analysis and Design
RNAP
IPTG
GFP
T7 GFPmetRpro
Unregulated Networks
• The metR promoter has been inserted between the T7 RNAP binding site and the GFP start site.
• The met operator in E.coli consists of tandem repeats of eight base pair sequences, homologous to a palindromic consensus AGACGTCT, known as “met boxes” to downregulate transcription.
• There are 4 met boxes in the metR promoter of E.coli. The sequences correspond to a 50%, 75%, 75%, and 63% identity to the consensus sequence.
AGgatTtT AGcCGTCc AGAtGTtT AcACaTCc
Robert Entus Motif Based Network Analysis and Design
Unregulated Networks
0
50000
100000
150000
200000
250000
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
IPTG (mM)
Flu
ore
sc
en
ce
/A6
00
• Curves where fitted to a simple Hill function using EasyGraph (Future Skill Software). Hill coefficients:
– T7 promoter = 1.93
– MetR promoter = 1.42
T7 promoter
MetR promoter
Robert Entus Motif Based Network Analysis and Design
T7 metJ
RNAP
IPTG
MetJ:metR promoter Network
• The met repressor is the product of the metJ gene. It is a stable homodimer in dilute solutions.
• The free repressor has a relatively low affinity for DNA. When it noncooperatively binds two molecules of SAM, with a Kd of 10-5 M, it forms an active repressor that has high affinity for DNA
• Met repressor makes direct contact with the major groove in the middle of the met box. This binding prevents RNAP read-through and decreases the amount of GFP produced.
Robert Entus Motif Based Network Analysis and Design
T7 metJ
RNAP
IPTG
GFP
T7 GFPmetRpro
MetJ:metR promoter Network
• The met repressor is the product of the metJ gene. It is a stable homodimer in dilute solutions.
• The free repressor has a relatively low affinity for DNA. When it noncooperatively binds two molecules of SAM, with a Kd of 10-5 M, it forms an active repressor that has high affinity for DNA
• Met repressor makes direct contact with the major groove in the middle of the met box. This binding prevents RNAP read-through and decreases the amount of GFP produced.
Robert Entus Motif Based Network Analysis and Design
T7 metJ
RNAP
IPTG
T7 GFPmetRpro
MetJ:metR promoter Network
• The met repressor is the product of the metJ gene. It is a stable homodimer in dilute solutions.
• The free repressor has a relatively low affinity for DNA. When it noncooperatively binds two molecules of SAM, with a Kd of 10-5 M, it forms an active repressor that has high affinity for DNA
• Met repressor makes direct contact with the major groove in the middle of the met box. This binding prevents RNAP read-through and decreases the amount of GFP produced.
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10 100 1000
IPTG (mM)
Rel
ativ
e F
luo
resc
ence
MetJ:metR promoter Network
AGgatTtT AGcCGTCc AGAtGTtT AcACaTCc 50% 75% 75% 63%
Native metR promoter
MetJ binding competitively inhibits RNAP. Resulting ing a decreasing amount of metR promoter-GFP hybrid transcription.
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10 100 1000
IPTG (mM)
Rel
ativ
e F
luo
resc
ence
MetJ:metR promoter Network
AGgatTtT AGACGTCT AGACGTCT AcACaTCc 50% 100% 100% 63%
AGgatTtT AGcCGTCc AGAtGTtT AcACaTCc 50% 75% 75% 63%
Native metR promoter
Mutated met promoter
Mutation of two met box sequences increases MetJ’s affinity, thereby increasing the amount of network repression present
Robert Entus Motif Based Network Analysis and Design
T7 antiGFP
GFP:antiGFP network
• Native regulatory RNAs have a wide variety of biological functions including the repression and activation of translation and the protection and degradation of mRNAs via base pairing with the target transcripts.
• Another group of small RNAs modifies protein activity by mimicking the structures of other nucleic acids.
• The antiGFP network utilizes the production of the reverse complement of the coding GFP mRNA as an inhibitor of GFP translation
• A T7 promoter downstream of the GFP coding region reading in such a way that the mRNA produced resulted in a reverse compliment of GFP including the -10 region (antiGFP)
RNAP
IPTG
Robert Entus Motif Based Network Analysis and Design
RNAP
IPTG
GFP
T7 GFP
T7 antiGFP
GFP:antiGFP network
• Native regulatory RNAs have a wide variety of biological functions including the repression and activation of translation and the protection and degradation of mRNAs via base pairing with the target transcripts.
• Another group of small RNAs modifies protein activity by mimicking the structures of other nucleic acids.
• The antiGFP network utilizes the production of the reverse complement of the coding GFP mRNA as an inhibitor of GFP translation
• A T7 promoter downstream of the GFP coding region reading in such a way that the mRNA produced resulted in a reverse compliment of GFP including the -10 region (antiGFP)
Robert Entus Motif Based Network Analysis and Design
RNAP
IPTG
GFP
T7 GFP
T7 antiGFP
GFP:antiGFP network
• Native regulatory RNAs have a wide variety of biological functions including the repression and activation of translation and the protection and degradation of mRNAs via base pairing with the target transcripts.
• Another group of small RNAs modifies protein activity by mimicking the structures of other nucleic acids.
• The antiGFP network utilizes the production of the reverse complement of the coding GFP mRNA as an inhibitor of GFP translation
• A T7 promoter downstream of the GFP coding region reading in such a way that the mRNA produced resulted in a reverse compliment of GFP including the -10 region (antiGFP)
Robert Entus Motif Based Network Analysis and Design
RNAP
IPTG
T7 GFP
T7 antiGFP
GFP:antiGFP network
• Native regulatory RNAs have a wide variety of biological functions including the repression and activation of translation and the protection and degradation of mRNAs via base pairing with the target transcripts.
• Another group of small RNAs modifies protein activity by mimicking the structures of other nucleic acids.
• The antiGFP network utilizes the production of the reverse complement of the coding GFP mRNA as an inhibitor of GFP translation
• A T7 promoter downstream of the GFP coding region reading in such a way that the mRNA produced resulted in a reverse compliment of GFP including the -10 region (antiGFP)
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10 100 1000
IPTG (mM)
Rel
ativ
e F
luo
resc
ence
GFP:antiGFP network
antiG
Partial length reverse compliment
antiGFP RNA binds GFP mRNA to competitively inhibits translation of measurable protein. Resulting in a decreasing amount of fluorescence.
Robert Entus Motif Based Network Analysis and Design
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10 100 1000
IPTG (mM)
Rel
ativ
e F
luo
resc
ence
GFP:antiGFP network
antiGFP
Full length reverse compliment
antiG
Partial length reverse compliment
Increasing the overall length of antiRNA transcript increases inhibitory effects.
Robert Entus Motif Based Network Analysis and Design
T7 Lyso
RNAP
IPTG
T7 RNAP:T7 lysozyme Network
• T7 lysozyme interacts with parts of the palm, finger, and the N-terminal domain of RNAP.
• Binding occurs in the cystosol in a DNA independent manner
• Protein flexibility is decreased, inhibiting a conformational change that is required to form a fully open initiation complex.
• Once the polymerase has cleared the promoter, the elongation complex (EC) is generally resistant to T7 lysozyme. However, if the RNA:RNAP interaction is disrupted the EC becomes sensitive to T7 lysozyme.
Robert Entus Motif Based Network Analysis and Design
T7 Lyso
RNAP
IPTG
GFP
T7 GFP
T7 RNAP:T7 lysozyme Network
• T7 lysozyme interacts with parts of the palm, finger, and the N-terminal domain of RNAP.
• Binding occurs in the cystosol in a DNA independent manner
• Protein flexibility is decreased, inhibiting a conformational change that is required to form a fully open initiation complex.
• Once the polymerase has cleared the promoter, the elongation complex (EC) is generally resistant to T7 lysozyme. However, if the RNA:RNAP interaction is disrupted the EC becomes sensitive to T7 lysozyme.
Robert Entus Motif Based Network Analysis and Design
T7 Lyso
RNAP
IPTG
GFP
T7 GFP
T7 RNAP:T7 lysozyme Network
• T7 lysozyme interacts with parts of the palm, finger, and the N-terminal domain of RNAP.
• Binding occurs in the cystosol in a DNA independent manner
• Protein flexibility is decreased, inhibiting a conformational change that is required to form a fully open initiation complex.
• Once the polymerase has cleared the promoter, the elongation complex (EC) is generally resistant to T7 lysozyme. However, if the RNA:RNAP interaction is disrupted the EC becomes sensitive to T7 lysozyme.
Robert Entus Motif Based Network Analysis and Design
T7 Lyso
RNAP
IPTG
T7 GFP
T7 RNAP:T7 lysozyme Network
• T7 lysozyme interacts with parts of the palm, finger, and the N-terminal domain of RNAP.
• Binding occurs in the cystosol in a DNA independent manner
• Protein flexibility is decreased, inhibiting a conformational change that is required to form a fully open initiation complex.
• Once the polymerase has cleared the promoter, the elongation complex (EC) is generally resistant to T7 lysozyme. However, if the RNA:RNAP interaction is disrupted the EC becomes sensitive to T7 lysozyme.
Robert Entus Motif Based Network Analysis and Design
T7 RNAP:T7 lysozyme Network
0
0.2
0.4
0.6
0.8
1
0.001 0.01 0.1 1 10 100 1000
IPTG (mM)
Fluo
resc
ence
T7 lysozyme concentration can be modulated to increase or decrease the overall amount of repression in the system
Low Lysozyme
Robert Entus Motif Based Network Analysis and Design
T7 RNAP:T7 lysozyme Network
0
0.2
0.4
0.6
0.8
1
0.001 0.01 0.1 1 10 100 1000
IPTG (mM)
Fluo
resc
ence
T7 lysozyme concentration can be modulated to increase or decrease the overall amount of repression in the system
Low Lysozyme
Medium Lysozyme
Robert Entus Motif Based Network Analysis and Design
T7 RNAP:T7 lysozyme Network
0
0.2
0.4
0.6
0.8
1
0.001 0.01 0.1 1 10 100 1000
IPTG (mM)
Fluo
resc
ence
T7 lysozyme concentration can be modulated to increase or decrease the overall amount of repression in the system
Low Lysozyme
Medium Lysozyme
High Lysozyme
Robert Entus Motif Based Network Analysis and Design
T7 Lyso
RNAP
IPTG
T7 GFP
RBS Mutations
• The lysozyme inhibitory network was mutated to decrease the amount of lysozyme present without changing RNAP kinetics.
• The ribosome binding site (RBS) was altered, from strong to weak, to decrease mRNA translation into active inhibitor.
• The ability of the T7 lysozyme to effectively inhibit GFP production is correlated with RBS Strength.
Robert Entus Motif Based Network Analysis and Design
T7 Lyso
RNAP
IPTG
T7 GFP
Ribosome Binding SiteMutated to modulate translation efficacy.
RBS Mutations
• The lysozyme inhibitory network was mutated to decrease the amount of lysozyme present without changing RNAP kinetics.
• The ribosome binding site (RBS) was altered, from strong to weak, to decrease mRNA translation into active inhibitor.
• The ability of the T7 lysozyme to effectively inhibit GFP production is correlated with RBS Strength.
Robert Entus Motif Based Network Analysis and Design
T7 Lyso
RNAP
IPTG
GFP
T7 GFP
Ribosome Binding SiteMutated to modulate translation efficacy.
RBS Mutations
• The lysozyme inhibitory network was mutated to decrease the amount of lysozyme present without changing RNAP kinetics.
• The ribosome binding site (RBS) was altered, from strong to weak, to decrease mRNA translation into active inhibitor.
• The ability of the T7 lysozyme to effectively inhibit GFP production is correlated with RBS Strength.
Robert Entus Motif Based Network Analysis and Design
RBS Mutations
0
0.2
0.4
0.6
0.8
1
1 10 100 1000
IPTG (mM)
Flu
ore
scen
ce
RBS Sequence TE*
1 AAGAAGGAGATATACCATG 1.0
* Translational efficiency
As the translational efficiency of the T7 lysozyme RBS decreases the overall amount of repression in the system is decreased as well.
Robert Entus Motif Based Network Analysis and Design
RBS Mutations
0
0.2
0.4
0.6
0.8
1
1 10 100 1000
IPTG (mM)
Flu
ore
scen
ce
RBS Sequence TE*
1 AAGAAGGAGATATACCATG 1.0
2 TAAGAAGGAAATTAATCATG 0.95
* Translational efficiency
As the translational efficiency of the T7 lysozyme RBS decreases the overall amount of repression in the system is decreased as well.
Robert Entus Motif Based Network Analysis and Design
RBS Mutations
0
0.2
0.4
0.6
0.8
1
1 10 100 1000
IPTG (mM)
Flu
ore
scen
ce
RBS Sequence TE*
1 AAGAAGGAGATATACCATG 1.0
2 TAAGAAGGAAATTAATCATG 0.95
3 AACACAGGAAAATTAATCATG 0.6
* Translational efficiency
As the translational efficiency of the T7 lysozyme RBS decreases the overall amount of repression in the system is decreased as well.
Robert Entus Motif Based Network Analysis and Design
RBS Mutations
0
0.2
0.4
0.6
0.8
1
1 10 100 1000
IPTG (mM)
Flu
ore
scen
ce
RBS Sequence TE*
1 AAGAAGGAGATATACCATG 1.0
2 TAAGAAGGAAATTAATCATG 0.95
3 AACACAGGAAAATTAATCATG 0.6
4 AACACAGGAACAATTAATCATG 0.45
* Translational efficiency
As the translational efficiency of the T7 lysozyme RBS decreases the overall amount of repression in the system is decreased as well.
Robert Entus Motif Based Network Analysis and Design
Conclusions• Three functional gene networks, based on a three gene feed forward loop
architecture, can be designed and built utilizing the inducible expression of T7 RNA polymerase.
• The presence of inhibition, not the macromolecular target of the inhibition, defines the network response. Three network designs, all based on different inhibitory elements produced biological concentration sensors, these include:
– T7 lysozyme inhibits RNAP through protein:protein interactions– AntiGFP binds mRNA transcribed by RNAP to prevent translation of measurable
GFP through RNA:RNA interactions– MetJ binds “met box” DNA sequences to prevent RNAP read through providing
protein:DNA inhibition• The network can be “tuned” to alter measurable characteristics to either shift
the apparent concentration of the peak or decreasing the inhibitory slope.• This work shows that it will be possible to design small modular networks
that could potentially be linked together to form larger networks that provide novel functionality