2. GFP emits by UV (364 nm) and the light is transferred to the other test tube through an optical fiber.
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mf.Lon+LVA tag is expressed and GFP is degraded by the Lon protease.

4. mf.Lon+LVA tag is degraded by ClpX from E.coli

We are trving to draw a movie of a firework that goes upward and bursts scattering light-emitting pieces. The basic green light sensor needs two plasmids.pPLPCB provides a light-sensing molecule that binds to CcaS molecule. pJT118 harbors the genes for CcaS and CcaR and the reporter lacZ gene under the control of the PcpcG2 promoter. CcaS activates　PcpcG2,when the light-sensing molecule catches green light, by activating phosphorylation of CcaR. We will substitute the lacZ gene with the GFP gene. Therefore, our green sensor should produce GFP in reaponse to green light.

We will draw, with E coli harboring pPLPCB and the GFP-substituted pJT118,a picture of a trajectory of a rising firework on an ager plate, and will “fire”with a pilot light at the bottom. Under the blue LED light(or weak UV light),the bottom part of the picture should begin to emit green light that should activate the neighboring bacteria. Because the GFP has a degradation tag, the emission will stop soon. It is exposed that, as a result, the green light will move upward along the trajectory until it reaches the top.

At the top, we put E.coli with pPLPCB and an RFP-substituted pJT118.This will sense the green light coming from the neighboring bacteria and emit red light.

At the last, we put E.coli with pPLPCB and an RFP-substituted pJT106b. This will sense the red light coming from the neighboring bacteria and emit red light.

With these plasmids, we believe that we could draw movie pictures on an ager plate canvas. Our first one would be that of a firework in which a ball of green light would go straight upward and burst, scattering lines of red light. The data for the plasmid construction experiments are shown below.

1.We amplified a sequence in pJT118 spanning almost all of the plasmid except the region of the lacZ ORF, by PCR with KOD Fx Neo. (using the primers GCGGCCGCTCGAGTCTAATTTTTTTG and ATCTATCATAGATAAAGTTAGTAATTAAAC). The 5780bp fragment was obtained and gel-purified.

2.We amplified the sequence of the GFP ORF from BioBrick(BBa-E0040) using the primers CTTTATCTATGATAGATATGCGTAAAGGAGAAGAACTT and GACCTGAGCGGCCGCTTTGTATAGTTCATCCATGCCAT.The 750bp fragment was gel-purified.

3.We put the fragment from 1and2 together by the InFusion svstem (Clontech). The miniprep plasmid sample from acolony was checked by agarose gel electrophoresis (Figure1,the middlelabeled as118+GFP). The other plasmids were constructed in almost the same way, and checked on agarose gels (Figures 1and 2).

We transformed the plasmids into JT2 (ΔEnvZ strain) and picked up an individual colony to put into liquid medium. Shaking incubate on 37℃ was continued for 42 hours. We expected that the E.coli under white light will express GFP or RFP, and on the other hand, the E.coli under Dark will not express them. Unfortunately, we were not able to watch that the E.coli having pJT118 (GFP and RFP) worked.

Purpose: search condition that expression RFP We used JT2 transformde plasmids(106b+RFP,cph8,pPLPCB) shaking incubate on37℃ 16 hours exposing white light. And it centrifuge, sprinkle M9 plate and incubate 24hours by three conditions (room temperature ).
1, red light
2, white light
3, dark
Result→ all condition expressed　RFP 目的 RFPが発現する条件を調べる。プラスミドを入れたJT2を使用。37度で白色光を当てながら浸透培養をしました。それを遠心機にかけ、大腸菌を集めてM9のプレートに撒きました。3つの条件で24時間培養しました。(温度はすべて室温) 1、赤い光を当てて 2、白色光をあてて ３、暗闇 結果　すべて発現した