Team:Tokyo Metropolitan/Project/Killing

From 2011.igem.org

(Difference between revisions)

Revision as of 17:56, 4 October 2011

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
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

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

Figure4: Killing Concept

@@ Line 45: / Line 45: @@
 </td></tr></table>
-</html>
 </p>
 <script src='http://partsregistry.org/cgi/partsdb/seq_edit/all.js'></script> <DIV id='sequencePaneDiv'> <INPUT type='hidden' id='new_dna_format' name='new_dna_format' value='' /> <INPUT type='hidden' id='selection_start' name='selection_start' value='14' /> <INPUT type='hidden' id='selection_end'   name='selection_end'   value='0' /></DIV> <script> var sequence = new String('cctctggtgactttatccgtaaataatttaacccactccacaaaaaggctcaacaggttggtggttctcaccaccaaaagcaccacaccccacgcaaaaacaagtttttgctgatttttctttataaatagagtgttatgaaaaattagtttctcttactctctttatgatatttaaaaaagcggtgtcggcgcggctacaacaacgcgccgacaccgttttgtaggggtggtactgactatttttataaaaaacattattttatattaggggtgctgctagcggcgcggtgtgtttttttataggataccgctaggggcgctgctagcggtgcgtccctgtttgcattatgaattactagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagtcacacaggaaagtactagatgccagaaaaacatgacctgttggccgccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgtttgcaatggcgtaccttcgcggcagatataatggcggtgcgtttacaaaaacagtaatcgacgcaacgatgtgcgccattatcgcctggttcattcgtgaccttctcgacttcgccggactaagtagcaatctcgcttatataacgagcgtgtttatcggctacatcggtactgactcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaagatggtagaaatcaataaatggtagaaatcaataatcaacgtaaggcgttcctcgatatgctggcgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatggttatgacgtcattgtaggcggagagctatttactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaatcaacaggcgccggacgctaccagcttctttcccgttggtgggatgcctaccgcaagcagcttggcctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagcagattaaggagcgtggcgctttacctatgattgatcgtggtgatatccgtcaggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctggttatggtcagttcgagcataaggctgacagcctgattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgaatgagcagagtcaccgcgattatctccgctctggttatctgcatcatcgtctgcctgtcatgggctgttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaaaaatgccagagaactgaagctggcgaacgcggcaattactgacatgcagatgcgtcagcgtgatgttgctgcgctcgatgcaaaatacacgaaggagttagctgatgctaaagctgaaaatgatgctctgcgtgatgatgttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccaccaccgcctccggcgtggataatgcagcctccccccgactggcagacaccgctgaacgggattatttcaccctcagagagaggctgatcactatgcaaaaacaactggaaggaacccagaagtatattaatgagcagtgcagatagtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata');        var seqFeatures = new Array( ['brick',445,1701,'K124017', 1], ['brick',1,355,'I714030', 1], ['brick',1710,1838,'B0015', 1], ['brick',1710,1789,'B0010', 0], ['brick',364,417,'R0040', 0], ['brick',1798,1838,'B0012', 0], ['brick',426,438,'B0032', 0], ['stop',1831,1831,'Stop', 0], ['reg',383,388,'-35', 0], ['reg',406,411,'-10', 0], ['rbs',426,438,'RBS-3\Weak', 0], ['rbs',432,435,'RBS', 0], ['wiggle',364,382,'TetR 1', 0], ['wiggle',389,407,'TetR 2', 0], ['h_pin',1721,1764,'', 0], ['h_pin',1805,1824,'T7 TE', 0], ['polya',1825,1838,'PolyA', 0]); var subParts = new Array (  new Part (10028, 'I714030', '', 'http://partsregistry.org/images/partbypart/icon_conjugation.png'),  new Part (187, 'R0040', 'p(tetR)', 'http://partsregistry.org/images/partbypart/icon_regulatory.png'),  new Part (149, 'B0032', '', 'http://partsregistry.org/images/partbypart/icon_rbs.png'),  new Part (12888, 'K124017', '', 'http://partsregistry.org/images/partbypart/icon_coding.png'),  new Part (603, 'B0010', '', 'http://partsregistry.org/images/partbypart/icon_terminator.png'),  new Part (145, 'B0012', '', 'http://partsregistry.org/images/partbypart/icon_terminator.png')); var Format = '_subparts_'; var PrimaryPartID = '23723'; var Selection_Start = 0; var Selection_End = 0; showSeqFeatures(false);  </script><div style='position:relative;clear:both;width:100%'><div style=''>
+</html>