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.

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 is from Vibrio fischeri: a transcription activator 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(?).

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.
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