robert entusmotif based network analysis and design synthetic biology network analysis and design...

Robert Entus Motif Based Network Analysis and Design

Synthetic Biology Network Analysis and Design

Robert Entus, Ph.D.


“Synthesis drives discovery and paradigm change in ways not possible through

analysis”

-Steve Brenner


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.


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.


Network SimplificationWithout simplification understanding network interactions becomes

very difficult

Example: Given b = 3.6 what does a =?

a4 + 4a3b + 6a2b2 + 4ab3 + b4 = 1296


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


AraC

O1O2 araI cI

cI

OR1 OR2 metJGFP

Arabinose

GFP

MetJ

Network For Feedback Amplifier


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?


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.


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


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


AraC

O1O2 araI cI

cI

OR1 OR2 metJGFP

Arabinose

GFP

MetJ

Working Model


AraC

O1O2 araI cI

AraC binds araI and O2, bending the DNA so that the two DNA bound AraC contact each other


AraC

O1O2 araI cI

cI

Arabinose

Arabinose causes a conformation change allowing AraC to bind araI and O1 allowing the transcription


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.


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.


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


AraC

O1O2 araI cI

cI

OR1 OR2 metJGFP

Arabinose

GFP

MetJ


metJ is transcribed from the same mRNA as GFP.


MetJ provides negative feedback in the system by preventing cI production.

AraC

O1O2 araI cI

cI

OR1 OR2 metJGFP

Arabinose

GFP

MetJ


MetJ induced negative feedback can be modulated in two different areas.

AraC

O1O2 araI cI

cI

OR1 OR2 metJGFP

Arabinose

GFP


The Ribosome Binding Site controlling MetJ translation.

Each 8 base sequence can be altered independently.


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


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.


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.


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.


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'


Polymerase Chain Reaction


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.


Network Assembly


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 -


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


lac promoter GFP

Ligation reaction

repeat process for additional genes


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


AraC

O1O2 araI cI

cI

OR1 OR2 metJGFP

Arabinose

GFP

MetJ

Network For Signal Linearization


AraC

O1O2 araI GFP

Arabinose

GFP



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


AraC

O1O2 araI

metJ

GFP

Arabinose

GFP

MetJ



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


AraC

O1O2 araI metJGFP

Arabinose

GFP

MetJ



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


AraC

O1O2 araI cI

cI

OR1 OR2 GFP

Arabinose

GFP



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


AraC

O1O2 araI cI

cI

OR1 OR2 metJGFP

Arabinose

GFP

MetJ



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


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


RNAP

IPTG







TAATACGACTCACTATA

RNAP

IPTG

T7 GFP







TAATACGACTCACTATA

RNAP

IPTG

GFP

T7 GFP







RNAP

IPTG

T7 GFP


• 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.


RNAP

IPTG

T7 GFPmetRpro






RNAP

IPTG

GFP

T7 GFPmetRpro








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


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.


T7 metJ

RNAP

IPTG

GFP

T7 GFPmetRpro






T7 metJ

RNAP

IPTG

T7 GFPmetRpro






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



Native metR promoter

MetJ binding competitively inhibits RNAP. Resulting ing a decreasing amount of metR promoter-GFP hybrid transcription.


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


AGgatTtT AGACGTCT AGACGTCT AcACaTCc 50% 100% 100% 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


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


RNAP

IPTG

GFP

T7 GFP

T7 antiGFP

GFP:antiGFP network






RNAP

IPTG

T7 GFP

T7 antiGFP

GFP:antiGFP network






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.


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.


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.


T7 Lyso

RNAP

IPTG

GFP

T7 GFP







T7 Lyso

RNAP

IPTG

T7 GFP








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



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


Low Lysozyme

Medium Lysozyme



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


Low Lysozyme

Medium Lysozyme

High Lysozyme


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.


T7 Lyso

RNAP

IPTG

T7 GFP

Ribosome Binding SiteMutated to modulate translation efficacy.

RBS Mutations





T7 Lyso

RNAP

IPTG

GFP

T7 GFP

Ribosome Binding SiteMutated to modulate translation efficacy.

RBS Mutations





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.


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*


2 TAAGAAGGAAATTAATCATG 0.95




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*



3 AACACAGGAAAATTAATCATG 0.6




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*



3 AACACAGGAAAATTAATCATG 0.6

4 AACACAGGAACAATTAATCATG 0.45




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


Acknowlegments

• Herbert Sauro (UW)• Vijay Chickarmane (Caltech)• V. Ravi Rao (KGI)• Frank Bergmann (UW)• Brian Aufderheide (KGI)

robert entusmotif based network analysis and design synthetic biology network analysis and design...

Documents

robert entusmotif

existing networks

molecular biology

small gene networks

complex gene networks

design robert entus

synthetic analogs

analysis steve brenner