Remote Control of Bacterial Chemotaxis

Remote Control of Bacterial Chemotaxis

UCSF iGEM Team 2006

Patrick Visperas

Matthew Eames

Eli Groban

Ala Trusina

Christopher Voigt, Tanja Kortemme, Chao Tang, Chris Rao

Remote Control of a Bacterial MachineRemote Control of a Bacterial Machine

Go

al

Sta

rt

How Bacterial Chemotaxis WorksBacteria Swim Up Gradients

concentration gradient

No gradient

CCW Glide

Tumble?CW

How Chemotaxis is Observed

Goulian Motility Assay (U Penn)

The Regulatory Network

WAP~

YP~

A

Y

No CheW

No Swimming

Binding Partners

•Orthogonal binding pair Tsr-CheW (Liu et al, 1991)

•We mapped these mutations onto the Tar-Chew complex

Tar

CheW

Tsr mutation can be mapped to Tar Gradient Aspartate

• point mutation in Tar* reduces motility to approximately 40% that of wild-type

Part Design• New orthogonal signaling pairs

“Codon randomization” is used to reuse scaffolds without fear of recombination

Chassis Engineering• Build E. coli strain lacking all sensors and CheW, but retaining the other chemotaxis proteins

tar tsr tap trg aer cheW

Wild-type E. coli

tar tsr tap trg aer cheW

UU1250 (Parkinson, U Utah)

UUcheW

tar tsr tap trg aer cheW

cheW* tar*

Tested in UU1250

FimE

IRL IRR

IRR IRL

OUTPUT[ara]

FimE

Ptrc*rfp gfp

MCS

GREEN

RE

D

0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 80 0.3 1.2 5[arabinose] (mM)

% b

act

eri

a

1. The plasmid is unstable

2. The strain contains FimE/ FimB

2 Possible Problems

Device Characterization: The Fim-Switch

- ara

+ ara

INPUT

T. Ham, et al (2006)

- arabinose

Phe Asp

araC PBAD fimE T1x4 CheW r2 Ptrc* r2 CheW* T1 PlacIQ r0 phe-tar r0 asp-tar*

Phe

CheYCheY

Constructed via DNA synthesis (DNA 2.0)

System Design (11 kB)

araC PBAD fimE T1x4 CheW r2 Ptrc* r2 CheW* T1 PlacIQ r0 phe-tar r0 asp-tar*

Asp

CheYCheY

Phe Asp

+ arabinose

Constructed via DNA synthesis (DNA 2.0)

- arabinose

Phe Asp

System Design (11 kB)

Results Gradient Gradient Aspartate Phenylalanine

Conclusions• Orthogonal pairs are a rapid method to built signaling pathways that can operate simultaneously

• Chassis developed to rapidly screen for new protein-protein interactions

• Limited by the switch performance in this chassis

• Directional control could also be achieved by non-chemical inputs (light, radiation, etc)