Increasing target specificity of a T6SS assassin strain.

Why induce tse2 expression only when a gram-negative pathogen is present?

A type six secretion system +/Tse2 +/Tsi2+ strain of E. coli could be administered like a traditional antibiotic after a pathogen infection by having a strain that constitutively expresses Tse2 and Tsi2. However, this would be a fairly brute force method of anti-bacterial comparable to traditional chemical antibiotics in its damage to the gut’s natural flora, and is only advantage over chemical antibiotics would be the current lack of Tse2 resistant pathogenic bacterial strains. If T se2 expression is induced by a chemical signal indicative of the presence of a pathogen, any unintended disruption of the natural gut flora will be limited to the area infected with pathogens, and the risk of Tse2 resistance would decrease. In addition, if Tse2 is only expressed when pathogenic bacteria are present, a Tse2+/ Tsi2+/ T6SS strain could be used in order to help prevent pathogenic gut infections, and could be a permanent member of the gut flora. Such a preventative strain could even be used against multiple gut pathogens by merely including multiple copies of tse2, each of which would respond to signals indicative of different pathogens.

3OC6HSL as a proxy for a pathogenic signal

Figure 1: Structure of 3OC6HSL

3-oxohexanoyl-homoserine lactone (3OC6HSL) is a small organic molecule produced by Vibrio fischeri as a quorum sensing signal. Quorum sensing is a pathway where every bacterial cell produces and excretes a specific signalling molecule. When a specific density of bacteria is present in a region ( a quorum), the level of quorum sensing molecules is sufficient to induce or repress specific genes in order to tune gene expression to an environment of high cellular density. In V. fischeri, 3OC6HSL permeates the cellular membrane and binds to the transcriptional factor LuxR, changing the conformation of LuxR in such a way that LuxR can bind to a DNA sequence within the Lux promoter, activating downstream transcription. While the 3OC6HSl-LuxR quorum sensing system is not present in any human pathogens, placing Tse2 downstream from a Lux promoter in a strain expressing LuxR does provide a good proof of concept showing that Tse2 can be regulated in such a way that Tse2 expression occurs only in the presence of a target bacterial pathogen( map of construct= figure 1, include). Many pathogenic bacteria do engage in quorum sensing. This system could be easily converted to express Tse2 only in the presence of a specific pathogenic species by changing the promoter and transcriptional factor. A major advantange of using the luxR quorum sensing system is that a well charactarized part containing both the LuxR transcriptional factor and Lux promoter already exists in the registry([http://partsregistry.org/Part:BBa_F2620 F2620]). This allowed for amplification from genomic DNA to be skipped, saving time.

Plan to use a F2620-Tse2 plasmid in Tsi2- cells to measure toxicity of Tse2 in 'E. coli'

Our initial plan was to tranform the F2620-Tse2 construct ( figure 2) into 'E. coli' cells not containing a copy of Tsi2. By increasing levels of 3OC6HSL in the culture medium of F2620-Tse2 construct + strains, one would expect to increase the amount of Tse2 transcription that would occur. By measuring relative cell growth in cultures containing increasing levels of 3OC6HSL ( this would be accomplished by measuring absorbance of liquid cultures at 600nm), one could get an indication of relative Tse2 level needed to cause 'E. coli' cell death. The toxicity of Tse2 could then be compared to that of other cellular toxins by replacing Tse2 in the F2620-Tse2 construct with other cellular toxins. Since culture 3OC6HSL levels is directly related to transcription from the Lux promoter, proteins that cause cell death only at higher 3OC6HSL are less toxic than proteins that cause cell death at lower 3OC6HSL concentrations. Characterization of toxicity of cell death proteins would allow for the correct choice of cell death protein for a given application.

Figure 2: Diagram of F2620-Tse2 construct