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Team:Uppsala
From 2014.igem.org
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Bactissiles: The future of microbial combat
Destabilized ecosystems and disturbed gut floras are both consequences of treatments that lack selectivity. More efficient and precise methods are needed. This year we, the Uppsala iGEM team, tries to widen the view and find new possibilities with engineered bacteria. By developing a system that homes towards a target and secretes an affectant, we can ensure a specific outcome. Such a system could have applications in a number of different fields, though we have chosen to put this into practice in a pinpointing pathogen-killing approach. In our prototype system, introduced in E. coli , we hijack the quorum sensing system of the gut pathogen Yersinia enterocolitica . Our bacteria will be able to sense the presence of the pathogen, accumulate in its vicinity and emit a target-specific bacteriocin, leaving the remaining gut flora intact. The era of mass destruction is over. Welcome the missile bacteria, the Bactissile! .
Assembly Plan
Main Result
The Sensing System
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By constructing the measurement construct BBa_K1381008 (yenbox_WT-B0032-GFP) and performing double transformation together with one of the constructs producting the activator YenR BBa_K1381005 (J23110-B0034-YenR), BBa_K1381006 (J23102-B0034-YenR) and BBa_K1381007 (J23101-B0034-YenR). We managed to show that the activator YenR works perfectly fine in E. coli and that it recognise the recognition region, the yenbox and induces the strength of the promoter fused with it. By measuring the production of the green fluorescence protein GFP using a flow cytometer, we could see that we got a five-fold induction when YenR with the strongest promoter out of the three used were present.
The Targeting System
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We managed to restore chemotaxis in a non-motilite mutant strain, by reintroducing the cheZ gene on plasmids into cheZ knock-out E. coli. The motility assay used to measure the chemotaxis was also improved by optimizing incubation conditions for cells inoculated in swarm plates. Three promoters of different strengths were tested in combination with our construct and were shown to induce different levels of motility, and hence different levels of cheZ.
The Killing System
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We manage to produce the bacteriocin colicin Fy and by fusing it with a His-tag we could peform a SDS-page and prove its presence. To analyse the colicin Fy’s killing efficiency we let Y. enterocolita grow with and without colicin Fy in liquid cultures and compared the result. Fig. 2 shows these two cultures plated. On the left plate, the Y. enterocolitica had grown with the colicin Fy added, while the right plate is the negativ control, where Y. enterocolitica had grown without any colicin Fy. If we compare the two plates, we can see that there is a clear difference in the amount of growing Y. enterocolitica colonies.
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