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Background

We wanted to use bacteriocin to design a new form of toxic that unlike general antibiotics, only kills specific pathogenes. Thereby leaving our gut flora intact. The goal was to express colicin in E.coli and also include an on and off system with sRNA together with the sensing system.

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 problem of antibiotic resistance spreading, we decided to use bacteriocins as our antimicrobial agent. A bacteriocin is a peptide that is produced naturally by certain bacteria and targets its close relatives. Unlike general antibiotics,the target of bacteriocins are very specific. The bacteriocin, colicin Fy is produced by Y. frederiksenii and it should mostly target other yersinia species. Bacteriocins will either attack the cell membrane or a mechanism inside the cell such as gene expression or protein production.^[1] One of colicins Fy main target is Y. enterocolitica. It kills Y. enterocolitica by creating pores in its cell membrane. Y. enterocolitica is also one among the common pathogens that infects the gut and cause some serious symptoms^[2]. These were some of the reasons to choose colicin as the bacteriocin we want to express.

Spot42 RNA

Our goal was to design a seek and kill system. This implies that we only want to express the bacteriocin when our bacteria is close to the target. We choose this strategy because the cell would stress out to express bacteriocin all the time, even when not in close proximity to Yersinia. In order to make our bacteria only express the colicin when we want, we have designed an sRNA system. We have based our design on the system that Team Uppsala iGEM 2012 implemented. .
The sRNA or spot42 consists of an antisense region that recognizes a specific RNA sequence to interact with and another region that recruits the protein Hfq. The Hfq protein blocks and prevents mRNA from binding to the ribosome. We have redesigned the antisense region so it will specifically recognize our USP45. This will make it bind to our USP45 and recruit the Hfq protein to stick on that region.^[3] 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. When our bacteria is in proximity of Y.entercolitica we want it to express the colicin Fy. Therefore, the sensing group have been working on a Yen system which will inactivate the promoter that regulates the spot42 when it is close to Y.enterocolitica.

Figure 2. The spot42 system

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[1] http://www.nature.com/nrmicro/journal/v11/n2/full/nrmicro2937.html
[2] http://jb.asm.org/content/194/8/1950.long
[3] https://2012.igem.org/Team:Uppsala_University/Project

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Team:Uppsala/Project Killing

From 2014.igem.org