Team:Tokyo Metropolitan/Project/Killing

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Killing Device

Concept
When bee attack enemy, they sting needle and inject toxin. In Japan, the most cause of death from animal is attacted from bee (especially wasp).Bee is awful animal but taking a hint from it, we propose new system for killing bacteria using conjugation.

Conjugation

Fig1.Conjugation[1]		E.coli has sex. “Male” E.coli (having conjugative plasmid) mate other “Female“ E.coli(not having conjugative plasmid). Male E.coli transfer conjugative plasmid to Female E.coli. Then, after conjugation, Female E.coli gets conjugative plasmid and change sex “male”.
Conjugation is useful system for killing bacteria. We plan to integrate killing gene (below-mentioned) into the conjugative plasmid and induce this plasmid into E.coli. Then E.coli conjugate other E.coli and send killing gene which induce E.coli to cell lysis when it expressed.

Cell Lysis
Bacteriophage have cell lysis cassette S,R,Rz called "Holin-Endolysin". S gene encodes "Holin" that forming hole on　inner membrane of host cell.
R gene encodes "Endolysin" degradates murein in intermediate membrane of host cell(in membrane of host cell,not harmful).Rz is also know for the enzyme attack outer membrane with R gene.
Using this system, we cannot only kill target bacteria which are conjugated but also be harmless for other organism. [[File:holin-endolysin.jpg]]
Anti-killer device
To prevent cell lysis of BeE.coli itself, anti-killing device is needed. We chose pTet-TetR system for repressing expression of Killing gene. TetR bind tet operator (TetO) and inhibit expression of tetracycline resistance gene. But in existence tetracycline, TetR formed complex with tetracycline and lost ability of binding tetO.
Then tetracycline resistance gene expressed. aTc (Anhydrotetracycline) is known as analog of tetracycline so aTc bind TetR like tetracycline and inhibit TetR to binding tetO.
Ptet-TetR system is appropriate Anti-killer Device. In BeE.coli, TetR is expressed constitutively and repressed expression of killing gene. But killing gene has sent to other bacteria which doesn’t have tetR. Then conjugated bacteria cell lysis. In addition, from point of view of biosafety, to add aTc induce BeE.coli to cell lysis.
Figure3: TetR is transcription regulator[3]


Figure4: Killing Concept


Parts Design
Killing Device
BBa_K543016

 Anti-killing Device
BBa_K543015


Result
Repress expression of GFP by Anti-killing device
We confirm whether Anti-killing Device is working or not. We transform Anti-killing Device into E.coli which have GFP generator(I13522:Ptet-B0034-GFP-B0015) and observe its florescence. Figure2 is a result of this experiment. Left is E.coli transformed Anti-killing Device, right is control. Although not perfectly repressed GFP expression, we confirmed Anti-killing Device is working. Figure5 is difference of GFP florescence by comparing with absorbance of 525nm. For perfectly repressed expression cording under Ptet, RBS is needed to be more strong(ex.B0034).In addition, we should do more experience to get quantitative date.
Figure5: repress expression of GFP by Anti-killing device
Sequence analysis
We check sequence of P0440(B0034-C0040-B0010-B0012).But sequence of C0040 was incorrect. Sequence of our P0440 is here.


Reference
[1]University of Birmingham, Horizontal gene transfer (HGT) in biofilms (http://www.birmingham.ac.uk/schools/biosciences/staff/profile.aspx?ReferenceId=6059)
[2]CHUNG-YU CHANG, KIEBANG NAM, AND RY YOUNG1. 1995.2 S Gene Expression and the Timing of Lysis by Bacteriophage λ. J. Bacteriol. 177: 3283–3294
[2]Maria A. Schumacher, Marshall C. Miller, Steve Grkovic, Melissa H. Brown, Ronald A. Skurray and Richard G. Brennan. 2002 Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR

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