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The assembly plan of the Targeting system.

Background

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 Fy 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 colicin Fy when our bacteria is close to the target. We choose this strategy because the cell would stress out to express colicin Fy all the time, even when not in close proximity to Yersinia. In order to make our bacteria only express the colicin Fy 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 Fy. The sRNA will stick to Hfq and bind to the mRNA of USP45-Colicin Fy 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

System design

The aim for the killing group was to construct two different constructs- one with an export tag and the bacteriocin, CFy, and the other with a sRNA system for inhibition of the toxin when not in proximity to yersinias. However, at first we wanted to insert our final construct into lactobacillus via a shuttle vector. Because lactobacillus is a probiotic we could use our system in a real scenario if you get an Yersinia infection in the gut. The final construct would consist of J23106-B0034-USP45-CFY and a construct for the sRNA spot42. The spot42 already includes an promtor.
Two different export tags were used. PelB for E.coli and USP45 for both Lactobacillus and E.coli. Two Anderson promoters were chosen, J23106 (strong) and J23116 (weak) for the colicin Fy because we wanted to find a balance, where E.coli would not die from stress (due to too high expression) or its own-produced toxin. To characterize our colicin Fy we wanted to purify our sample so we could see the protein on SDS-Page. In order to do this a His-Tag was required for an IMAC column purification. Therefore we also built the constructs with a 6X His-Tag.

We designed the colicin Fy according to the Freiburg assembly standard, biobrick 25. This would allow us to fuse our protein with the export tag without having to consider the stop codon from the scar of SpeI and XbaI from the biobrick 10 standard.

Silencing RNA system design

The silencing RNA system or sRNA system was also one of the primary aims for the killing group. We designed and modified the sRNA system from Team Uppsala iGEM 2012 called spot42. The spot42 was redesigned to sense our USP45 sequence and bind to it. We call our modified part, Spot42_USP45. The Spot42_USP45 system consist of an antisense region that will recognice our USP45 and it also has the part that recruits the Hfq protien that will block and inhibit the mRNA translation. The Hfq protein will stick to the antisense region, this will block the RBS from binding to the mRNA therefore inhibits the translation. We have designed three different antisense regions for our Spot42_USP45. They are all based on USP45 but with different lengths, 10, 15 and 20bp. This is to ensure that it is specific to only USP45. Otherwise it could bind to another region and inhibit a process that might be vital for the bacteria. We choose to have USP45 as our primary export tag since our initial goal was to build our system in lactobacillus. To help us with that we used knowledge from the Uppsala iGEM 2013 who worked with lactobacillus and USP45 among other things.

The whole system design

Our two constructs the spot42_USP45 and J23106-B0034-USP45-CFY was to be coupled with the parts from the other project groups. When our bactosile is close to Y.enterocolitica the YenR system will sense the OHHL molecule.This will inhibit the yenbox system and thereby inhibit the CheZ and Spot42_USP45. This will make the bacteria stop and since the Spot42_USP45 is inhibited, the colicin Fy will be produced and it will be in attack mode. And when the bactisile is not in proximity of the OHHL the yenbox system will be active and it will produce CheZ and Spot42_USP45 and make the bactisile motile and not producing any colicin Fy making it be in tracking mode. See figure 3.

Result

Characterization

Our initial plan was to have an secretion-tag together with our colicin Fy allowing it to secret from the living cells. Unfortunately we did not manage to get the assembly of any of the constructs to fully work. Our enzymes for the Freiburg standard did not seem to work so we had to use SpeI and XbaI causing a stop-codon that we did not managed to mutagenise away.

We had however succeeded in creating construct without secretion-tag. The expression of colicin Fy with x6 HIS-tag was characterized via an purification of the protein with IMAC followed by an SDS page to confirm lenght, [Link to protocol]. 11 tubes of eluent from the IMAC was saved. The protein concentration in each tube was analyzed with a nanodrop. Tube 4 and 6 had higher concentration than the other tubes and was run on SDS page.

The results from the characterization experiments can be seen in figure 4 and 5. According to calculations based on the nucleotide sequence, the mass of colicin Fy is about 49,6 kDa. The protein ladder we used is Thermo Scientifics PageRuler Unstained Protein Ladder #26614 (figure 3). The thickest band is 50 kDa so our bands should be about at the same height.

	Figure 3: Thermo Scientifics PageRuler Unstained Protein Ladder #26614
	Figure 4: The gel contains the following from the left to the right 1. Protein ladder 2. Eluent 1 for lysate produced by bacteriocin producing bacteria 3. Eluent 3 for lysate produced by bacteriocin producing bacteria 4. Eluent 2 for lysate produced by bacteriocin producing bacteria 5. Final eluent test-tube 4 for lysate produced by bacteriocin producing bacteria 6. Final eluent test-tube 6 for lysate produced by bacteriocin producing bacteria 7. Eluent 2 for lysate produced by the negative control 8. Eluent 3 for lysate produced by the negative control 9. Final eluent test-tube 4 for lysate produced by the negative control 10. Final eluent test-tube 6 for lysate produced by the negative control

Parts

• [1] (2013) "Bacteriocins — a viable alternative to antibiotics?" Paul D. Cotter et al
• [2] (2012) "Novel Colicin FY of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation" J.Bosak et al
• [3] (2012) https://2012.igem.org/Team:Uppsala_University/Project