Transcript IGEM2006-UCSF-Powerpoint

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
Goal
Remote Control of a Bacterial Machine
Start
How Bacterial Chemotaxis Works
Bacteria Swim Up Gradients
How Chemotaxis is Observed
Goulian Motility Assay (U Penn)
Glide
CCW
CW
concentration gradient
No gradient
?
Tumble
The Regulatory Network
A W
P~
No CheW
A
No Swimming
P~
Y
Y
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
UU1250 (Parkinson, U Utah)
Wild-type E. coli
tar tsr tap trg aer
cheW
tar tsr tap trg aer
cheW
UUDcheW
cheW*
tar*
tar tsr tap trg aer
cheW
Device Characterization: The Fim-Switch
INPUT
[ara]
OUTPUT
MCS
Ptrc*
rfp
IRR
IRL
gfp
FimE
FimE
IRL
IRR
T. Ham, et al (2006)
Tested in UU1250
35
30
2 Possible Problems
25
1. The plasmid is
unstable
20
15
2. The strain contains
FimE/ FimB
10
5
RED
+ ara
% bacteria
- ara
0
0
0.3
1.2
5
1 2 3 4 5 6 7 8
GREEN
[arabinose] (mM)
System Design (11 kB)
Phe
CheY
araC PBAD
fimE
T1x4
CheW r2
Ptrc*
r2 CheW* T1 PlacIQ r0 phe-tar r0 asp-tar*
Constructed via DNA
synthesis (DNA 2.0)
Phe
Asp
- arabinose
System Design (11 kB)
Asp
CheY
araC PBAD
fimE
T1x4
CheW r2
Ptrc*
r2 CheW* T1 PlacIQ r0 phe-tar r0 asp-tar*
Constructed via DNA
synthesis (DNA 2.0)
Phe
Asp
- arabinose
Phe
Asp
+ arabinose
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 proteinprotein interactions
• Limited by the switch performance in this chassis
• Directional control could also be achieved by nonchemical inputs (light, radiation, etc)