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The assembly plan of the Killing 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 1. The spot42 system

System design

The aim for the killing group was to construct two different constructs - one with a secretion tag and the bacteriocin, colicin Fy and the other with a sRNA system for inhibition of the toxin when not in proximity to Y. enterocolitica. However, at first we wanted to insert our final construct into Lactobacillus via a shuttle vector. Since Lactobacillus is a probiotic we could use our system in a real scenario if you get a Y. enterocolitica infection in the gut. The final construct would consist of J23106-B0034-USP45-CFY and a construct for the sRNA spot42. The sRNA spot42 already includes an promtor.
Two different secretion 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 a 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.

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 2 and 3. 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 2). The thickest band is 50 kDa so our bands should be about at the same height.

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

Several IMAC and SDS pages were run, see read more. In all cases eluent 1 was found to be empty. This is not surprising because the eluent volume is so small that the proteins will not come out until eluent 2. Because of this we decided to skip eluent 1 for the negative control in the presented SDS in figure 3. Eluent 2 is consists of several proteins, every protein that does not bind to the IMAC should come out in this step. Eluent 3 seems to be when our colicin Fy is eluted, seeing it shows a band at the correct length, 50kDa. The negative control did not show any band in 50kDa and expression could therefore be confirmed. Since the colicin was eluted at elution 3 no band can be seen at elution 4.

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Appendix 1 displays an earlier SDS-page that was run with more constructs. Since there were more construct we could not try all elutes, since they did not fit on one gel. The existence of colicin Fy in the construct with J23106 could be seen though, due to this we continued to try further characterization with this construct (SDS-page gel before).

Appendix 1: The gel contains the following from the right to the left: 1. Protein ladder 2. Final eluent 1 with CP 11 promoter 3. Final eluent 2 with CP 11 promoter 4. Final eluent 1 with J23106 promoter 5. Final eluent 2 with J23106 promoter 6. Final eluent 1 with J23113 promoter 7. Final eluent 2 with J23113 promoter 8. Final eluent 1 with DH5-alpha as negative control 9. Final eluent 2 with DH5-alpha as negative control 10. Stage 3 eluent with promoter J23106

A in the appendix 1 is pointing towards the 50 kDa band on the ladder. B is pointing towards the band that can be seen and that likely is the colicin Fy while C is another eluent with the same promoter also showing the colicin Fy existence. This shows that the promotor J23106 can be used to produce the colicin, while CP 11 and J23113 is not.

We did manage to get some indication that the CP11 promoter might have worked but the band was not strong enough to guarantee that it was actually there. There can be quite a few reasons to why the band is not stronger than it is. There is a possibility that the protein was eluted out of the system too early which also seems to be the case to some extent. Appendix 1 lane 3 shows a band in the right position, this was not observed for the other tests but since that eluent is in a falcon tube containing about 40 ml of liquid it was probably very diluted and weak. It could also be some leakage from the well next to it.

The IMAC and SDS-page managed to provide a strong indication that we did produce the colicin Fy. There existed bands at the right places and the negative control did not have any bands at all. All experiments were conducted in the same manner and if it was another protein that by chance bond to the IMAC gel it should have been in the negative control as well. Most bacteriocins are not fully studied so there might always be unknown problems with them. One possibility is that they somehow hurt the bacteria in which they are produced in. Since the colicin described here is supposed to only react to a specific receptor that some Yersinia species have on the cell surface the probability for the colicin to hurt the producing bacteria is probably very low. A larger problem could be that many bacteriocins are short lived in certain conditions and might denature or break before they are characterized.

Inhibition test

To be able to prove that our system is working we needed to test it on the actual Y. enterocolitica. Since Y. enterocolitica is a class two bacteria we had to do it in another lab. We managed to get in contact with Livsmedelsverket (the Swedish authority of food safety) and they allowed us to test our colicin Fy in one of their labs on one of their pathogenic strains of Y. enterocolitica.

To examine the inhibition of colicin Fy a overnight culture of 200ml with no antibiotic was prepared. As negative control DH5-alpha was grown under the same conditions. No antibiotic was added to ensure that the antibiotics would not affect the results.The cells were then lysed and the supernatant was collected according to protocol. Supernatant was added to a final concentration of 1%(v/v) in LB with 10^4 CFU/ml Y. enterocolitica added.. The culture was put to grow in 37d C with 100rpm shaking. OD measurements were taken every hour, but due to Y. enterocolitica ability to aggregate the results were inconclusive. Instead 100microL was plated on BHI agar plates after 0h, 2h, 4h and 24h. Immediately growth difference could be spotted and after 4 hours the results were very clear, figure 4. More figures of the plating can be found in the appendix.

Figure 4: Tests to compare colicin Fy lysate on the right plate compared to the N/C on the left, both plates are diluted to the same concentration as taken at the same time, for more figures check the "Read more"

Colicin Fy specificity test

To see if the Colicin Fy really is as specific as we thought, we had to try it on other bacteria. Apart from testing them on Y. enterocolitica we also managed to get a hold of risk class one versions of Pseudomonas, Enterobacter, Klebsiella as well as Lactobacillus plantarum and the standard E. coli DH5-alpha. We did multiple tests with varying results. The test were performed in the same way as the inhibition test above. However OD600 measurements were made here since no aggregations could be seen in most of our cultures, see table A. The table indicates that the colicin Fy is probably specific, but something seems to have gone wrong with the plating, see figures E, and that result was inconclusive.

OD value after 2H

OD value after 4h

OD valueafter 24H

*The broth used to grow the lactobacillus bacteria wasn´t available to calibrate the spectrophotometer with. Therefore the values will be wrong but the comparison betwen the control and the colicin Fy should still be valid.
**The bacteria had created precipations which makes the measurings unreliable.

Since the experiment did not give any clear results we did the experiment again with new lysates, one with colicin Fy and one negative control. At first it seemed as if the colicin Fy killed most of them but at a closer look we saw two types of colonies on some of the plates, probably contamination. When plating out the lysates that should be sterile, some bacteria still grew, but they grew much more in the controls. If the contaminated plates are ignored the colicin Fy seems specific, but more experiments need to be done to be sure. Due to lack of time this was not possible.

As seen in figure 7 the control lysate is much more contaminated than the bacteriocin, this explains why the control plates in all cases have more bacteria on the plates plated with 300 µl of lysate added compared with all the others. Different types of colonies can also be seen on most of them.
In conclusion there are some indications that colicin Fy is specific but more tests need to be done.

Read more/Hide

Appendix 2: The K on the plates stands for klebsiella, the C stands for control lysate added while B for bacteriocin (colicin Fy) added. the numbers 10, 100 and 300 stands for the amount of µl of lysate added.

Appendix 3: The D on the plates stands for DH5-alpha (E. coli), the C stands for control lysate added while B for bacteriocin (colicin Fy) added. The numbers 10, 100 and 300 stands for the amount of µl of lysate added.

Appendix 4: The P on the plates stands for pseudomonas, the C stands for control lysate added while B for bacteriocin (colicin Fy) added. The numbers 10, 100 and 300 stands for the amount of µl of lysate added.

Parts

Fav.	BioBrick code	Type	Construct	Description	Designers
	BBa_K1381014	Regulatory	J23101-spot42_USP45_10bp	Silencing sRNA with affinity to USP45	Killing Group
	BBa_K1381015	Regulatory	J23101-spot42_USP45_15bp	Silencing sRNA with affinity to USP45	Killing Group
	BBa_K1381016	Regulatory	J23101-spot42_USP45_20bp	Silencing sRNA with affinity to USP45	Killing Group
	BBa_K1381017	Coding	CFY-X6 his	The gene coding for the bacteriocin colicin Fy	Killing Group
	BBa_K1381018	Tag	B0034-USP45	The secretion tag USP45 with the RBS B0034	Killing Group
	BBa_K1381019	Device	B0034-pelB-CFY-X6 his	The bacteriocin colicin Fy coupled to the the secretion tag pelB	Killing Group
	BBa_K1381020	Generator	J23106-B0034-pelB-CFY-X6 his	Colicin Fy coupled to the the secretion tag pelB with the promoter J23106	Killing Group
	BBa_K1381021	Generator	J23116-B0034-pelB-CFY-X6 his	Colicin Fy coupled to the the secretion tag pelB with the promoter J23116	Killing Group
	BBa_K1381022	Device	B0034-USP45-CFY-X6 his	The bacteriocin colicin Fy coupled to the the secretion tag USP45	Killing Group
	BBa_K1381023	Generator	J23106-B0034-CFY-X6 his	Colicin Fy coupled with the promoter J23106	Killing Group

• [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