From 2014.igem.org

(Difference between revisions)

Revision as of 20:37, 13 October 2014

Home

Failed to load tracking. JS is probably not enabled

Background

We wanted to use bacteriocin to design a new form of toxic that unlike general antibiotics, only kills specific pathogenes. Thereby leaving our gut flora intact. The goal was to express the bacteriocin colicin Fy in E.coli that is specific against Y.enterocolitica a pathogene found in the gut. We also wanted to include an on and off system with silencing RNA together with the sensing system.

System design

From the Yen system we stole the activator YenR and the recognition region, the yenbox, fused with a wild type promoter. YenR was synthesized with the RBS B0034 and codon optimized for E. coli. A customized version of the yenbox-promoter was also created.

Result

We managed to prove the existance of the bacteriocine on a SDS-page. By adding lysate that contains the colicin Fy to living yersinia cultures we could also show that it works for killing the actual pathogen as well.

Parts

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

Parts

[1] http://www.nature.com/nrmicro/journal/v11/n2/full/nrmicro2937.html
[2] http://jb.asm.org/content/194/8/1950.long
[3] https://2012.igem.org/Team:Uppsala_University/Project

Back to Top

@@ Line 31: / Line 31: @@
 <h2 class="overview">Result</h2>
-<p class="box_text">We managed to prove the existance of the bacteriocine on a SDS-page. By adding lysate that contains the bacteriocin to living yersinia cultures we could also show that it works for killing the actual pathogen as well.
+<p class="box_text">We managed to prove the existance of the bacteriocine on a SDS-page. By adding lysate that contains the colicin Fy to living yersinia cultures we could also show that it works for killing the actual pathogen as well.
 </p>
 </a>

Team:Uppsala/Project Killing

From 2014.igem.org

Revision as of 20:37, 13 October 2014

Home

Background

System design

Result

Parts

Assembly Plan

Background

Bacteriocin

Spot42 RNA

System design

Silencing RNA system design

The whole system design

Result

Characterization

Read more

Parts