Note: the content on this page is possiblybeing moved into the intro, and the sections on recombineering the T6SS and cloning a tse2/tsi2 system

Using Type VI Secretion as an Antibacterial Agent

The T6SS we want to use has several difficulties in its current form. First, it is found in P. aeruginosa which can be pathogenic, as well as not being an enteric bacteria. As our model is anti-enteric, the native system would be unfeasible.

Secondly, T6SS is highly regulated at both the transcriptional and post-transcriptional level. We want our toxin delivery system to be expressed in either a constitutive or easily inducible manner.

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Heterologous Expression of T6SS

In order to create a probiotic application for this system, we first attempt to express it heterologously in non-pathogenic E. Coli. Starting from a Fosmid containing our T6SS, we are using [http://web.ncifcrf.gov/research/brb/recombineeringInformation.aspx Recombineering] to replace the strict native regulation with robust T7 promoters to create strong expression of the T6SS.

All the essential genes for our T6SS are contained within two putative operons, encoded in opposite directions. The native promoters for both operons are found in the same intergenic region, between fha1 and tssA1. Therefore, we can easily replace the promoter regions for both operons in one step.

Toxin/Antitoxin Inducible Circuit

One of the major proteins exported into prokaryotic cells by the T6SS of is the toxin Tse2 ( type six excreted 2). Tse2 is toxic to a wide range of gram-negative bacteria. In P. aeruginosa, Tse2 is coexpressed on the same operon with Tsi2. Tsi2 binds to Tse2 until Tse2 is excreted by the T6SS, and acts as an antitoxin. Inducing transcription of the Tse2/Tsi2 operon initiated the ability of the T6SS to cause cell death ( puncturing of the cellular membrane with the T6SS does not cause cell death). By inducing Tse2 production only when a pathogen is present, T6SS engineered bacteria would be a more finely targeted antibacterial agent than traditional chemical antibiotics.