Overall Objective:Using Type VI Secretion as an Antibacterial Agent

Our overall goal is to clone the Type 6 secretion system and the Tse2/Tsi2 toxin/antitoxin system from Pseudomonas aeruginosa into E. coli to make a strain of E. coli that could be used to kill off gram negative pathogens present in the human gut. Ideally, this system would be regulated in such a way that the strain of E. coli would only be able to kill gram negative bacteria when a gram negative pathogen is present. This strain could (ideally) be introduced into the gut either as a preventive measure or as a treatment after a known infection. Ideally, the probiotic would only kill of gram negative bacteria in the area of infection.

Cool Picture goes here

Designing the T6SS for a E. coli probiotic

Justin and or Laura, please write this section short intro, basic plan: -molecular schematic of T6SS (Jesa send to Matthew) -include map of, describe T6SS putative operon ( there is a map color coded to -mention that it is in pao1 and state objective overall: transfer into ecoli so that it functions constitutively , what genes

===Objectives:

1. figure out which genes are necessary

Mention not all genes in operon neccessarily needed for T6SS, preferable to be smaller

2. physically transfer genes into E. coli

mention large size makes it difficult, mention/explain fosmid, include fosmid map ( Laura should have This)

3. optimize regulation

mention that fosmid contains natural promoter, may not be trascribed from at all, or at right amount for a plasmid ( more than 1 copy of plasmid) 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.

Design of a Tse2/Tsi2 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.