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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
Sensing Result
Graph 2. The production of the green fluorescence protein GFP in cells containing the following constructs:
1. pSB3C17-B0032-yenbox_WT-GFP
2. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23101-B0034-YenR
3. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23110-B0034-YenR
4. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23102-B0034-YenR
By constructing the measuremnt 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.
Targeting Result
The perpose of the targeting system is to ensure that our Bactissile will be as close to the pathogen as possible upon producing the killing bacteriocin. We chose to achieve this by controlling the Bactissiles chemotaxis through the protein CheZ. The targeting module is connected to the sensing module, which induces a lower production of CheZ when the Bactissile is closer to the pathogen, causing the Bactissile to stop. This way, we hoped to be able to control chemotaxis.
After successfully assembling a promoter, an rbs and cheZ, we did manage to restore chemotaxis in one test by inserting this construct in a cheZ knock-out. However, since this only happened in one isolated instance, this is not evidence enough to prove the success of our construct, however it does indicate that through more rigorous and consisted testing more reliable results could be obtained. We therefore consider to have proved that our system could work, although at this moment we don’t have any definitive proof.
Killing Result
The goal of this years project is to kill the gut pathogen Yersinina enterocolitica. this will be done by using the bacteriocin colicin FY. The plan was to fuse the bacteriocin with an export-tag but due to failures in assembling in the end we had to do our characterization by lysating our cells and take the bacteriocine from that supernatant. Doing this we were able to make a SDS-page and show the bacteriocins existance and further on adding the lysate to living yersinia culures. Seen in the picture are 2 plates of Y.enterocolitacas, the left one grown in a liquid culture with bacteriocine added while the other one is a negative control. There is a clear diference in the amount of yersinias so we can then show that the colicin is a succsess.
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