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Introduction

The kill switch mechanism is based on the properties of the colicin protein. A colicins are a type of toxin proteins produced by and toxic to some strains of Escherichia coli. Colicins are produced by bacteria to reduce competition from other bacterial strains. They do so by binding to outer membrane receptors, and using them to translocate to the cytoplasm or cytoplasmic membrane via penocytosis. Once inside, they exert their cytotoxic effect on the cytoplasmic membrane (depolarisation), DNase activity, RNase activity, and more.

-Cascales E, Buchanan SK, Duche D, Kleanthous C, Lloubes R, et al. Colicin biology. Microbiol Mol Biol Rev. 2007;71:158–229.

The pathway works via a two plasmid system: the first plasmid is responsible for sensing an environmental stimulus, the second one is responsible for creating the colicin to lyse the cells in the environment. Therefore, the first plasmid must trigger the next one to work. This is done in this experiment by creating a LuxI protein in the first plasmid. This protein is able to create diffuse-able acyl-homoserine lactones (AHLs) which are naturally involved with quorum sensing bacterial communication mechanisms. However, the second plasmid uses the AHL transcriptional receptors to create the colicin protein. As a result, the kill switch is engaged and results in non discriminatory cell death.

-Parsek MR, Val DL, Hanzelka BL, Cronan JE, Jr, Greenberg EP. Acyl homoserine-lactone quorum-sensing signal generation. Proc Natl Acad Sci U S A. 1999;96:4360–4365.

Step 1: PcstA+RBS+LuxI+double terminator

PcstA is a glucose repressible promoter system. Low glucose concentration results in increased activity by adenylate cyclase. cAMP binds to the cAMP receptor protein, which, in its bound form, is able to associate with the promoter and promote transcription of the LuxI downstream gene. The LuxI is an enzyme that creates acyl-homoserine lactones (AHL) from normal cell metabolites. All LuxI proteins direct the synthesis of specific AHLs and show sequence similarity.

Registry source: LuxI description source: http://partsregistry.org/wiki/index.php?title=Part:BBa_C0061

http://partsregistry.org/Part:BBa_J23039

Step 2: AHL Inducible Colicin E2 with GFP

This part is inducible with AHL to fluoresce green and activate the colicin genes. Specifically, there is a LuxR receptor which is activated by homoserine lactones and results in transpiration of downstream genes such as Colicin. This protien is a type of bacteriocin produced by and toxic to some strains of Escherichia coli such as strain BL21.