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Team:Chiba/Project:Sandbox
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Chiba 2009 Project: A Bacterial Timer
Introduction
- our project is to make a "bacterial timer" i.e. ~~(ぐたいてきに).
- our approach to this goal is to make a series of a transcription factor which each of them differes in a responce time(?) of the transcription activation(?) by an single(same?) inducer.
- we believe that this device would be useful for making an macroscopic(?) timing control(?) in bacterial behavior or many application in synthetic biology & iGEM community.
- to demonstrate this "timing control", we aimed(?) to draw an "animated picture": a picture that pop up (emerge?) one by one.
Project Design
- Our project is to make an series of a transcription factor which will be activated by an same inducer, but differs in the responce time.
- to achive this goal, we choosed a trascription factor LuxR, and tried to make a "delayed"-LuxR mutants by directed evolution, and tested its performance.
LuxR
- LuxR is from Vibrio fischeri: a protein is used in Quorum Seinsing.
- when LuxR binds to N-acyl homoserine lactone (AHL), LuxR-AHL complex is dimerilized and then activate the transcription below(?) the lux box promoter(?).
Experiments
Directed Evolution of LuxR to achive a "delayed" mutant
- We mutated LuxR gene by error-prone PCR using Mn2+(Joyce, 1996) and cloned it into a pUC vector.
- This library was transformed into XL10-Gold, and the plasmid was mini-prepped to get an plasmid library (pUC-[luxR]).
- The plasmid library was transformed into XL1-Blue harboring pAC-plux-gfp, which will glow green when an active LuxR was present.
- We screened for the fast/dull mutant of LuxR, and collected the clone's plasmid.
- ....
