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 Holin-Endolysin. Holin forms hole on outer membrane of host cell.
Endolysin is degradation peptide glycan in intermediate membrane of host cell.	Figure2 Bacteriophage Lysis Casstte[2]

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 we can’t confirmed correct sequence but, instead of P0440, we checked this sequence is partial (100% homology).

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

@@ Line 16: / Line 16: @@
 <tr><td><u><b><font size="3">Conjugation</font></b></u><br>
-<table><tr><td width="300" align="center"><img src="https://static.igem.org/mediawiki/2011/5/55/Tokyo_Metropolitan_conjugation.jpg" width="250"><br>Fig1.Conjugation</td><td width="500">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”.
+<table><tr><td width="300" align="center"><img src="https://static.igem.org/mediawiki/2011/5/55/Tokyo_Metropolitan_conjugation.jpg" width="250"><br>Fig1.Conjugation[1]</td><td width="500">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”.
 <br>
 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.</td></tr></table>
@@ Line 24: / Line 24: @@
 <tr><td><u><b><font size="3">Cell Lysis</font></b></u><br>
 <table><tr><td colspan="2">
-Bacteriophage have cell lysis cassette Holin-Endolysin. Holin forms hole on outer membrane of host cell.</td></tr><tr valign="top"><td width="400">Endolysin is degradation peptide glycan in intermediate membrane of host cell.</td><td><img src="https://static.igem.org/mediawiki/2011/f/f1/Tokyo_Metropolitan_Lysis.jpg" width="400"><br><div align="center">Figure2 Bacteriophage Lysis Casstte</div></td></tr></table>
+Bacteriophage have cell lysis cassette Holin-Endolysin. Holin forms hole on outer membrane of host cell.</td></tr><tr valign="top"><td width="400">Endolysin is degradation peptide glycan in intermediate membrane of host cell.</td><td><img src="https://static.igem.org/mediawiki/2011/f/f1/Tokyo_Metropolitan_Lysis.jpg" width="400"><br><div align="center">Figure2 Bacteriophage Lysis Casstte[2]</div></td></tr></table>
 </td></tr>
@@ Line 33: / Line 33: @@
 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. <br></td></tr><tr><td width="400">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. <br>
 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.</td><td width="400"><img src="https://static.igem.org/mediawiki/2011/e/ee/Tokyo_Metropolitan_Anti-killer.png" width="398"><br>
-<div align="center">Figure3: TetR is transcription regulator</div></td></tr></table>
+<div align="center">Figure3: TetR is transcription regulator[3]</div></td></tr></table>
 <br>
 </td></tr>