remote control of bacterial chemotaxis ucsf igem team 2006 patrick visperas matthew eames eli groban...
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![Page 1: Remote Control of Bacterial Chemotaxis UCSF iGEM Team 2006 Patrick Visperas Matthew Eames Eli Groban Ala Trusina Christopher Voigt, Tanja Kortemme, Chao](https://reader036.vdocument.in/reader036/viewer/2022062308/56649d135503460f949e772f/html5/thumbnails/1.jpg)
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
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Remote Control of a Bacterial MachineRemote Control of a Bacterial Machine
Go
al
Sta
rt
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How Bacterial Chemotaxis WorksBacteria Swim Up Gradients
concentration gradient
No gradient
CCW Glide
Tumble?CW
How Chemotaxis is Observed
Goulian Motility Assay (U Penn)
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The Regulatory Network
WAP~
YP~
A
Y
No CheW
No Swimming
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Binding Partners
•Orthogonal binding pair Tsr-CheW (Liu et al, 1991)
•We mapped these mutations onto the Tar-Chew complex
Tar
CheW
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Tsr mutation can be mapped to Tar Gradient Aspartate
• point mutation in Tar* reduces motility to approximately 40% that of wild-type
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Part Design• New orthogonal signaling pairs
“Codon randomization” is used to reuse scaffolds without fear of recombination
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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*
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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)
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- 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)
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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)
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Results Gradient Gradient Aspartate Phenylalanine
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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)