E. coli Time ManagerChiba University iGEM Team 2009

Undergraduates : Yoshimi Iyama, Kaoru Yamamoto, Makoto NozawaAdvises : Kei Terakubo, Yohei Tashiro, Maiko Furubayashi, Hiroki Fukutomi, Masahiro TominagaInnstractors : Prof. Daisuke Umeno, Prof. Taro Toyota, Prof. Kyoichi Saito

Implementing a "Timer" Function!・Our project is to make a sort of "timer", where gene activation is triggered after a certain time.・Our approach to this goal is to create a series of transcription factors (TFs) that can be activated by the same inducer molecule but with a different sensitivity. In an environment where the inducer concentration gradually levels up, these TFs switches on one by one according to the order of sensitivity.・We believe such collection of such TF variants would be useful for the timing control in biological function at will, and thereby contribute to the synthetic biology & iGEM community.

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a picture that pop up one by one.

Experiments, Results & Discussion

・Using a set of TF variants, we aimed to draw an "animated picture": a picture that pop up one by one.

Project Design・To create such a timer, we utilized LuxR, a protein used to mediate cell-cell communication during quorum sensing, as shown in the figure on the right.・By inserting point-mutations in the luxR gene, we sought to create LuxR proteins requiring a variety of lengths of time to activate downstream transcription.

GFPuv

pLuxI

LuxR

pUC

RR

RR R R

AHL Function of LuxR :(1)LuxR protein generation, (2)AHL binding domain, (3)dimerization, (4)DNA binding domain

cell

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Characterization of LuxR Mutants

JW1226 bacterial strains co-transformed with plasmids containing either wild-type or mutant LuxR and pLux-GFP were cultured. Those colony was then transfered to a solid medium containing 0, 1, 10, 100, or 1000 nM AHL. We observed fluorescence through the naked eye under UV light of 365nm.

Making Delayed-LuxR Mutants We created a mutant library of LuxR with the use of error-prone PCR and incorporated the resulting coding region into expression vectors.

The vectors were then transformed into a strain of E.coli JW1226 cells harboring plux-gfp. The colonies were lifted off the agar plate with a nitrocellulose filter and transfered to a platecontaining AHL in order to observe the evolution of GFP fluorescence over time. We selected 13 colonies that were slow to display fluorescence,which we designated as delayed-LuxR mutants.

Time-course color development�on the plate with fixed (100 nM) conc.

Demonstration

Genotypes

conclusion Reference

Fluorescence scored�at near-end point (6h)

Mutant LuxRs showed different threshold in AHL concentration required for switching.

・#2 and #4 : Similar threshold value of [AHL] with WT. ・Most others : Required about 10x higher concentration for switching. ・#1, #12, and #13 : Required as much as 100x higher concentration of AHL (1uM).

・#1 and #2 : As sensitive to AHL as wildtype but less efficient in activating the Lux promoter, thereby realizing the apparent delay in our system.・Many others : Exhibited slower color development, and they did not reach the maximum level of fluorescence.

The following lists where point-mutations were incorporated into the luxR gene.

A delayed GFP expression (in comparison to wild-type LuxR) phenotype was observed from colonies housing genes in which either the AHL-binding domain or the DNA binding domain had been mutated.

・By using error-prone PCR, we have created a LuxR library.・With a simple and convenient screening method, we have isolated various LuxR mutants which confer delayed switching behavior in GFP signals.・By introducing these LuxR variants together with reporter genes (such as GFP) under the control of Lux promoter, we created bacteria 'ink's that develop their color with unique delay-time.・We conducted painting with these bacteria inks thereby created animated pictures.・Characterization of LuxR mutants is ongoing. But our preliminary data showed that some of the variants turned out to be the one with less sensitivity to AHLs, and others seemed to be as sensitive as wild-type LuxR but seemed less efficient somewhere in the downstream process.

T = 0h 2.1h 2.5h 3h 3.8h 4.3h 5h 6h

We conducted painting with these bacteria inks thereby created animated pictures. The follw picture is "E. coli Timer"!!

(1)R.G.Zhang et al.: nature 417,2002,971-974.(2)Melissa B. Miller and Bonnie L. Bassler.: annurev.micro.55.1.165.2001 (3)W. nasser et al.: Anal. Bioanal. Chem. 387,2007,381-390.

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