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Assembly Plan

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

Colicin Fy is a bacteriocin that should specifically target Yersinia Entrecolitica and could potentially work as an alternative to modern day antibiotics. During the summer of 2014, Uppsala iGEM have worked to make the colicin into a working alternative to antibiotics and then characterize it. The bacteriocin was synthesized with a Histidine tag so that we would be able to extract it with IMAC and then prove its existence with the help of SDS-page. The first step in preparing for IMAC is to grow 200 ml liquid cultures that gets lysated by sonification. [LÄNK TILL PROTOKOL] The histidine tag on the end of the protein has affinity towards the nickel ions in the gel in the IMAC (Immobilized metal affinity chromatography). When the Histidine tag gets stuck in the column it is possible to filter out most other proteins that does not have high enough affinity towards the gel and thereby we hopefully end up with only the bacteriocin. [länk till protokol] It is not possible to control if the result from the IMAC is correct on its own but needs to be considered together with the result from the SDS-Page gel. The end product from the IMAC was 10-11 tubes (as seen in picture 1) for each bacterial strain containing 2 ml of liquid each. Other eluents in the steps before were saved in order to be able to see if the protein was eluted in an earlier stage than the final stage. Analyzing the protein concentration in all tubes resulted in that tube 4 and 6 usually had higher concentration than the other tubes and were therefore taken to the next step. The first step in preparing for IMAC was to grow 200 ml liquid cultures that was lysated by sonification. [LÄNK TILL PROTOKOL]Usually IMAC is also done with a pump but since the system is fairly small, gravitation was enough in this experiment. 2 columns were used in the experiments, that way it was possible to pour the buffers into the one situated higher up without disrupting the gel in the second column as well as giving a steady flow.

Picture 1: End product from the IMAC runs, each row contains eluents from the same run distributed between 10-11 ependorph tubes.	Picture 2. The IMAC and how it was assembled

SDS-page

After having purified the protein with the help of IMAC the next step is to show that it is actually there. SDS-page in theory works like a normal gel electrophoresis but it is for proteins instead of nucleic acid. Proteins get a similar charge and structure due to the substances SDS, DTT as well as heating it to 95 degrees, centigrade for 5 minutes and thereby it is possible to distinguish them by size alone. By having a protein ladder next to it, it is possible to see what size the other proteins in the gel are.

Eluent 1 is empty, this might at first be surprising but the volume added is so smal that the free proteins not sticking will not come out until eluent 2. because of this we decided to skip eluent 1 for the negative controll. Eluent 2 is comprised of lots of different proteins, everything not sticking to the gel should come out in this step. there was an error in the loading of the samples so eluent 2 and 3 have changed place compred to what is locigal. Eluent 3 seems to be when our colicin Fybacteriocin was eluated seeing it showed a band at the correct length. The negative controll did not show any band in this spot. Seeing the protein was eluated here there wasn´t anything left to get out on the higest concentraion.

The IMAC and SDS-page managed to provide a strong indication that we did produce the colicin Fybacteriocin. There existed bands at the right places and the negative control did not have any bands at all. All the experiments were conducted in the same manor and if it was another protein that by chance stuck to the IMAC gel it should have been in the negative control as well. Regarding the different promoters we used J23106 ,is a well-studiedwell studied promoter and it is the one that is the clearest that it is actully there.

We did managed to get some indication that also the CP11 promoter might also have worked but the band wasn´t strong enough to be sure. There can be quite a few reasons to why the band isn´t stronger than it is. Apart from the histidine tag posibly having an inhibiting efect Tthere is also a possibility that the protein was eluted out of the system too early which also seems to be the case to some extent. FigurePicture 5 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 coulds also only be some leakage from the well next to it.

Most Bacteriocins aren´t fully studied so there might always be unknown troubles with them, one possibility is that they somehow hurt the bacteria in which they areit is produced, in. Bbut since the colicin described here is supposed to only react to a specific receptor that some Yersinia. E haves on the cell surface the chance for that is probably very low. A bigger problem could be that many Bacteriocins are short lived in certain conditions and might denature or break before they are characterized. Another factor that might interfere with the expression of the protein is that according to some articles a Histidine tag at the C-terminal can inhibit the expression as much as to only produce a tenth of the original production level.

Colicin Fy specificity test

To se that Colicin Fy realy is as specific as we though we had to try it on other bacterias. We Apart from testing them on Y. enterocoliticas we also managed to get ahold of clas 1 versions of pseudumonas, enterobacter, klebsiella as well as lactobacilus plantarum and the standard E. coli DH5-alpha. We did multiple test with varying results. During the first experiment apart from plating the cultures we also measured the absorption of liquid cultures at OD value 600. The results can be seen in the table below, it indicates that the colicin Fy is probably specifik but something seems to have gone wrong with the plating and the result is inconclusive.

OD value after 2H

	DH5-alpha	pseudomonas	enterobacter	klebsiella	lactobacillus
Controll	0.390	0.085	0.620	0.563	1.100*
Colicin Fy	0.378	0.098	0.618	0.578	0.109*

OD value after 4h

	DH5-alpha	pseudomonas	enterobacter	klebsiella	lactobacillus
Controll	0.520	0.137	0.639	0.669	1.747*
Colicin Fy	0.503	0.139	0.658	0.748	1.815*

OD valueafter 24H

	DH5-alpha	pseudomonas	enterobacter	klebsiella	lactobacillus
Controll	1.003**	1.903	1.151**	1.012	2.589**
Colicin Fy	1.464**	1.990	1.825**	1.035	2.540*

*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.

Livsmedelsverket (Food administration)

To be able to prove that our system is working we needed to test it on the actual dangerous bacteria. Since Y.entrecolitica is a class two2 bacteria we had to do it in another lab. We managed to get in contact with Livsmedelsverket (the Swedish autorithy of food safety) and they allowed us to test our colicin Fybacteriocin in one of their labs.

Our initial plan was to have an export-tag together with our colicin Fybacteriocin allowing it to come out from the living cells. Unfortunately we didn´t manage to get any of the constructs to fully work. We designed the colicin Fybacteriocin 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 standards. Oour enzymes did not seem to work however so we had to use SpeI and XbaI causing a stop-codon that we did not managed to mutagenise away.

What we did manage to do though was, as seen from the SDS-page, to show that the colicin Fybacteriocin was actually expressed. To get the colicin Fybacteriocin out of the cell we lysate it by sonication [ länk till protokol!!! ] and after that centrifugedcentrifugated it at max speed to get rid of cell organelles and other bigger things. To see that it truly is the colicin Fybacteriocin, we grew two2 cultures under the same conditions and then added the same amounts and concentrations of them to liquid cultures. After that we plated them during different times to compare the differences. All tests showed that the colicin Fybacteriocin always killed more yesrsinas than the negative controll.

picture 7: Tests to compare colicin Fybacteriocin 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 picture check the APENDIX?

Parts

	BioBrick code	Type	Construct	Description	Designers
	BBa_K1381014	Regulatory	J23101-spot42		Killing Group
	BBa_K1381015	Regulatory	J23101-spot42		Killing Group
	BBa_K1381016	Regulatory	J23101-spot42		Killing Group
	BBa_K1381017	Coding	CFY-X6 his		Killing Group
	BBa_K1381018	Tag	B0034-USP45		Killing Group
	BBa_K1381019	Device	B0034-pelB-CFY-X6 his		Killing Group
	BBa_K1381020	Generator	J23106-B0034-pelB-CFY-X6 his		Killing Group
	BBa_K1381021	Generator	J23116-B0034-pelB-CFY-X6 his		Killing Group
	BBa_K1381022	Device	B0034-USP45-CFY-X6 his		Killing Group
	BBa_K1381023	Generator	J23106-B0034-CFY-X6 his		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