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Team:Tokyo Tech/Project/M13 Shuriken
From 2013.igem.org
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[1]Atsushi Higashitani. Nahoko Higashitani. Kensuke Horiuchi, DNA Replication in Filamentous Bacteriophage, Protein, Nucleic Acid and Enzyme(1994), vol39:2189-2197. | [1]Atsushi Higashitani. Nahoko Higashitani. Kensuke Horiuchi, DNA Replication in Filamentous Bacteriophage, Protein, Nucleic Acid and Enzyme(1994), vol39:2189-2197. | ||
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[6]H Nojiri Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System Biosci, Biotechnol, Biochem, 76(1), 1-18, 2012 | [6]H Nojiri Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System Biosci, Biotechnol, Biochem, 76(1), 1-18, 2012 | ||
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Revision as of 19:47, 24 September 2013
Abstract
E.ninja throws "shuriken" to attack E. samurai in response to C12-AHL cell-cell communication signal. In our story, M13 phages are the shuriken. Using plaque forming assay, we confirmed AHL-dependent release and infection of M13 phage shuriken.
Phage life cycle dependent on g2p expression
In natural M13 phage life cycle, g2p expression is required for production of single stranded DNA (ssDNA) and thus required phage release. Nicking on RF (replication form) phage DNA by G2p enzyme activity triggers a series of reactions to amplify the phage ssDNA. A phage coat proteins are then assembled around the ssDNA. [1][2]
Inducible M13 phage release
in the whole circuit design
In our story, E. ninja releases the phage particles, only when E. ninja is in the attack state induced by cell-cell communication molecule. In the whole circuit (Fig1), g2p expression is indirectly regulated by C12-AHL via CI expression.
Model system for inducible phage release
In this project, we constructed a model system for inducible phage release by regulation of g2p expression. Genome DNA of this engineered phage, shown in Fig2, needs two functions. One is inducible expression of g2p protein. We thus designed to replace the promoter for g2p protein with plux promoter. Note that we used C6-AHL, not C12-AHL, in this model experiment. The other is maintenance of the genome DNA in the absence of g2p expression. We designed to combine M13 genome RF DNA with pSB3K3 backbone.
Firstly, we confirmed that M13 genome with two modifications (PlacIq BBa_I14032) related to our design kept plaque forming activity. One is replacement of the promoter for g2p protein with a constitutive promoter, pLacIq(BBa_I14032). The other is accommodation of pSB3K3 backbone. Even though the plasmid has 2 different types of replication origins, M13 origin and pSB3 origin, this plasmid (BBa_K1139020) formed plaque. In contrast, construction intermediates without a promoter for g2p coding sequence (Promoterless-M13 + Plac, Promoterless-M13 + PlacGFP BBa_K1139022) could not form plaque. (See more about our plasmid construction:最下層へ)
We then confirmed that replacement of the g2p protein with plux accomplished inducible phage release.
Application
