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Background

In our project we developed a system to fight pathogens in the gut, which is not a unique idea. Except we wanted our systems key attribute to be specificity. To do so, we needed to find a unique quorum sensing language that we could hijack and utilize. We found the Yen system, from Yersinia enterocolitica. A homologous sytem to the well known Lux system, from Vibrio fischeri.

System design

From the Yen system we stole the activator YenR and the recognition region, the yenbox, fused with a wild type promoter. YenR was synthesized with the RBS B0034 and codon optimized for E. coli. A customized version of the yenbox-promoter was also created.

Result

We managed to get a five-fold induction relative to the base level of expression. The cells that were used during the measurements were double transformed with the yenbox-promoter complex with GFP and a plasmid producing the activator YenR.

Parts

Assembly Plan

Figure 1. The assembly plan of the killing construct. The green arrow is the promotor, the red circle is the RBS and the yellow box is the gene which consist of an export tag, USP45 and the bacteriocin, colicin FyA fused together.

Bacteriocin

With the growing problems of antibiotics we decided to use bacteriocincs as our antimicrobial. A bacteriocin is a peptide that is produced naturally by certain bacteria. It target close relatives to the producing bacteria. Unlike antibiotics the targets of bacteriocins are very specifc. The bacteriocin, colicin Fy is produced from y. frederiksenii and it will only target other yersinia species. Bacteriocins will either attack the cellmembrane or within the cell such as gene expression or protein production. One of colicins Fy main target is the y.enterocolitica. It kills y. enterocolitica by creating pores in its cellmembrane. Y. enterocolitica is also one of the more common pathogens in the gut and it causes some serious symptoms. That is why we choose colicin as the bacteriocin we want to express.

Spot42 RNA

Our goal was to design a seek and kill system. This means that we only want to express the bacteriocins when our bacteria is close to the target. We choose to do this because it takes energy to express the bacteriocins and there’s no use in overproducing it when the bacteria is not in proximity to y.entercolitica. In order to make our bacteria too only express the colicin when we want, we have included a sRNA system. We have an antisense region of the spot42 to recognize the USP45. USP45 is the secretion tag that we have coupled to the colicin. The sRNA will stick to Hfq and bind to the mRNA of USP45-Colicin gene and that will block the translation.
But when our bacteria is in proximity to y.entercolitica we want it to start express the CFyA. Therefore the sensing group have been working on a yenbox system which will inactivate the promotor that regulate the spot42 when it is close to y.enterocolitica.

Figure 2. The spot42 system

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Team:Uppsala/Project Killing

From 2014.igem.org