ECOLITASTER: Cellular Biosensor

Valencia iGEM 2006

Outline

Introduction Parts Design Systems Design Experimental work Conclusions

“To have success in science, you need some luck. Without it, I would never have become interested in genetics”. J.D. Watson.

Introduction

Objectives: Design a genetic system consisting on few genes that is

able to give a graded response according to a concentration of an input.

Modular project. Different devices. Use a biological mechanism to connect the membrane

receptor with the genetic network, obtaining a cellular biosensor.

Use new synthetic parts.

Project Design

This project is formed by two devices: a sensor and an actuator.

We use OmpR-P as input in order to assemble them.

We use a vanillin receptor (design in silico) as a sensor.

Our genetic circuit (actuator) is based on Weiss’ group work (Basu, Nature 2005) in order to obtain a graded response versus the concentration of a given input.

Incoherent circuit. Semi-digital interpretation.

RFP & GFPVanillin

Actuator Behavior

CRP

cI

RFPOmpR

GFP

tetR

CRP

cI

RFPOmpR

GFP

tetR

CRP

cI

RFPOmpR

GFP

tetR

[Vanillin]

P

P

P

V

V

V

Vanillin Receptor: mechanism

OPEN CLOSED

SIGNAL

Specificitydesign

Independentof stimulus

90R

164E

105N

89K

15D16N

214D

235E

103N

pdb 2DRI271 res

V

PPBP GN

E. coli

Parts Design

Promoters are critical elements designing those networks.

We focus our interest in binary promoters, i.e., promoters regulated by two transcription factors.

Integrating two signals. Reduce the number of genes of the circuit. Small size device. Different implementations exhibiting logic behaviors, but

not necessarily. Computational protein design.

Vanillin Receptor: DESIGNER methodology

First pre-compute all possible pair interactions for later use

iiij

ji

ij

ij

ij

ijASA

r

qq

r

b

r

aE

.612

foldedfoldedfolded

foldΔG

Folded: Fixed backbone + rotamerlibrary. Pairwise interactions, G ≈ E

unfoldedunfoldedunfolded

EG (AA)U

CHARMM22

Combinatorial problemAt each position we consider all rotamers R for each aminoacid a a1

a2a3R1

R2

R..

R3

82 4

53

9

Stability

Bin

din

g

Stabilityregion

Bindingregion

Pareto setnon-dominated

solutions

Stability

Bin

din

g

Stabilityregion

Bindingregion

Pareto setnon-dominated

solutions

vdw elec solbat

Systems Design

System and expected behavior. Model and simulations. Sensitivity analysis. Robustness analysis. Our biological system.

System and Expected Behavior

CRP

cI

RFPOmpR

GFP

tetR

CRP

cI

RFPOmpR

GFP

tetR

CRP

cI

RFPOmpR

GFP

tetR

[Vanillin]

P

P

P

V

V

V

Model and Simulations

We use an effective model, modeling only protein concentrations:

We consider generic parts to make these simulations. Thus, we take common values for the parameters from the literature. However we expect a similar behavior:

γ+β[Y]K[U]+

α=[Y]dtd

n -/1

1

Sensitivity Analysis

The well working of the circuit depends on the promoters upstream of the two branches: pOmpR and pOmpRm.

Robustness Analysis

We study the robustness of the gene circuit when there are oscillations in the sensing device. To perform that, we introduce a white noise in the input (OmpR-P).

OmpR-P OmpR-P OmpR-P

RFP

GFP

RFP

GFP

RFP

GFP

time time time

Our Biological System

Experimental Work

Parts construction. Where are the parts?

Repositories. E. coli genome. Built from scratch.

Making our BioBricks. pAND. Vanillin receptor. Fusion protein.

FACS results. Our Registry.

Where are the parts? (I)

Repositories:

pOmpR pOmpRm pLac pTetR GFP RFP TetR cI Tar-EnvZ

Where are the parts? (II)

E. coli genome: Trg CRP

Where are the parts? (III)

Built from scratch: pAND Vanillin PBP

Making our BioBricks (I)

pAND: pAND

CRP BS cI BS

-93,5 -42XbaI

[Joung, Science 1994]

Making our BioBricks (I)

pAND:

5’

5’

3’

3’

F0 F32 F71

R0R16R51R91

pAND

CRP BS cI BS

-93,5 -42XbaI

[Joung, Science 1994]

Making our BioBricks (I)

pAND:

5’

3’ 5’

3’

DNA ligase

5’

5’

3’

3’

F0 F32 F71

R0R16R51R91

pAND

CRP BS cI BS

-93,5 -42XbaI

[Joung, Science 1994]

Making our BioBricks (I)

pAND:

5’

3’ 5’

3’

DNA ligase

5’

5’

3’

3’

F0 F32 F71

R0R16R51R91

DNA polimerase & R91 + F71PCR

5’

3’ 5’

3’

5’

3’ 5’

3’

5’

3’ 5’

3’

pAND

CRP BS cI BS

-93,5 -42XbaI

[Joung, Science 1994]

Making our BioBricks (II)

Vanillin receptor:aa sequence:

KDTIALVVETLNKPDNVSLKDGAQKEADKLGYNLVVLDSQNNPAKELANVQDLTVRGTKILLIVPTDSDAVGNAVKMANQANIPVITLKRQATKGEVVSHIAADNVLGGKIAGDYIAKKAGEGAKVIELQGKAGTSAARELGEGFQQAVAAHKFNVLASQPADEDRIKGLNVMQNLLTAHPDVQAVFAQQDEMALGALRALQTAGKSDVMVVGDVGTPDGEKAVNDGKLAATIAELPDQIGAKGVETADKVLKGEKVQAKYPVDLKLVVKQ

pBSKValencia

$$ or €€

Computational design:Combinatory optimization

DESIGNER

Making our BioBricks (III)

NdeItrg

NdeItar envZ

Fusion protein Trz. [Baumgartner, J. Bact. 1993]. chemoreceptor Trg: periplasmic and transmembrane

domains. osmosensor EnvZ: cytoplasmic kinase/phosphatase

domain.

Making our BioBricks (III)

BioBrick PCR

Genomic PCR

NdeItrg

NdeItar envZ

Fusion protein Trz. [Baumgartner, J. Bact. 1993]. chemoreceptor Trg: periplasmic and transmembrane

domains. osmosensor EnvZ: cytoplasmic kinase/phosphatase

domain.

Making our BioBricks (III)

NdeI digestion

NdeI digestion &

dephosphorilation

BioBrick PCR

Genomic PCR

NdeItrg

NdeItar envZ

Fusion protein Trz. [Baumgartner, J. Bact. 1993]. chemoreceptor Trg: periplasmic and transmembrane

domains. osmosensor EnvZ: cytoplasmic kinase/phosphatase

domain.

Making our BioBricks (III)

NdeI digestion

NdeI digestion &

dephosphorilation

mix + ligate

BioBrick PCR

Genomic PCR

NdeItrg envZ

NdeItrg

NdeItar envZ

Fusion protein Trz. [Baumgartner, J. Bact. 1993]. chemoreceptor Trg: periplasmic and transmembrane

domains. osmosensor EnvZ: cytoplasmic kinase/phosphatase

domain.

Making our BioBricks (III)

NdeI digestion

NdeI digestion &

dephosphorilation

mix + ligate

BioBrick PCR

Genomic PCR

NdeItrg envZ

NdeItrg

NdeItar envZ

Fusion protein Trz. [Baumgartner, J. Bact. 1993]. chemoreceptor Trg: periplasmic and transmembrane

domains. osmosensor EnvZ: cytoplasmic kinase/phosphatase

domain.

Making our BioBricks (IV)

From wild type to BioBrick, a powerful screening method:

S PpTetR-RFP E X

Trg-envZ S P E X

Making our BioBricks (IV)

From wild type to BioBrick, a powerful screening method:

S PpTetR-RFP E X

Trg-envZ S P E X

EcoRI + PstI digestion & dephosphorilation

EcoRI + PstI digestion

Making our BioBricks (IV)

From wild type to BioBrick, a powerful screening method:

S PpTetR-RFP E X

Trg-envZ S P E X

EcoRI + PstI digestion & dephosphorilation

EcoRI + PstI digestion

mix & ligate &

transformation

Making our BioBricks (IV)

From wild type to BioBrick, a powerful screening method:

S PpTetR-RFP E X

Trg-envZ S P E X

EcoRI + PstI digestion & dephosphorilation

EcoRI + PstI digestion

mix & ligate &

transformation

pTetR-RFP

trg-envZ

FACS results (I)

Promoter pOmpR with GFP as reporter:

Negative control:XL1-Blue

Positive control:Green fluorophore

Set: pOmpR-RBS-GFP-T

FACS results (II)

Characterization of pOmpR and pOmpRm.

Negative control:XL1-Blue

Positive control:Green fluorophore

Set: pOmpR-RBS-GFP-T

Set: pOmpR-RBS-GFP-T

Our Registry

Parts submited by Valencia:New Parts:

pOmpR + RBS-GFP-T

(J 58102)

pOmpRm + RBS-GFP-T (J 58103)

pAND(J 58100)

pAND + RBS-RFP-T (J 58101)

PBP vanillin sensor (J 58105)

Fusion protein Trg-EnvZ(J 58104)

Conclusions

We have designed a genetic system consisting on 7 genes, expected to give a graded response according to vanillin concentration.

We use the phosphorilation mechanism to connect the membrane receptor with the genetic network, obtaining a cellular biosensor.

Our use of a two-regulator promoter allows to integrate signals and reduce the number of genes required for a device.

Computational design of a PBP-vanillin receptor.

Acknowledgements

EU FP6 NEST SYNBIOCOMM project (financial support).

Escuela Técnica Superior de Ingenieros Industriales (Universidad Politécnica de Valencia).

Instituto de Ciencia Molecular (Universitat de València).

E. O’Connor (FACS services). A. Moya and A. Latorre (Cavanilles).

UPV-UV Valencia iGEM 2006

Our team