remote control of bacterial chemotaxis
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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)
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