Difference between revisions of "Part:BBa K1381017"

Revision as of 18:59, 17 October 2014

CFY-X6 his

Colicin Fy
The CFY is a bacteriocin in the colicin family. It is produced naturally by Y.frederiksenii and target close relatives to that bacteria. One among them is the Y.enterocolitica, which is our target pathogen. The colicin Fy will target the cellmembrane of it's target.

The killing system
We designed the killing system to secret the colicin Fy with the help of a secretion tag, the USP45 BBa_K1381018. This part will be without promoter, RBS and export tag. It only contains the colicin Fy and a 6x His-tag for characterization. It is designed so that you can easily couple another promoter or export tag of your choice to it. This part contains the restriction sites NgoMIV at the start of the sequence to more easily fuse proteins like an export tag to it. Since it got a NgoMIV site at the start of the sequence it is possible to fuse other sequences if they follow the Freiburg Biobrick standard 25 and contains an AgeI site. However our part is not entirely built in the Freiburg standard Biobrick 25 since it does not contain an AgeI site.

Characterization
To be able to characterize colicin Fy we had to couple it to a promoter and an RBS, which we did by creating the construct BBa_K1381023. To be able to see that colicin Fy was really produced we needed to run an SDS-Page. Before running the SDS-page we needed to extract colicin Fy from the inside of the cells by sonication and then purify the tests so that we only have our protein colicin Fy. Since the bacteriocin was designed with a histidine tag we could use an IMAC to to do this. After we had done this we could run the SDS-page gel.

[http://2014.igem.org/Team:Uppsala/Project_Killing#ref_point2 link to more results]

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Figure 1: The gel contains the following from the left to the right
Protein ladder
Eluent 1 for lysate produced by colicin Fy producing bacteria
Eluent 3 for lysate produced by colicin Fy producing bacteria
Eluent 2 for lysate produced by colicin Fy producing bacteria
Final eluent test tube 4 for lysate produced by colicin Fy producing bacteria
Final eluent test tube 6 for lysate produced by colicin Fy producing bacteria
Eluent 2 for lysate produced by the negative control
Eluent 3 for lysate produced by the negative control
Final eluent test tube 4 for lysate produced by the negative control
Final eluent test tube 6 for lysate produced by the negative control

The results from the characterization experiments can be seen in figure 1. 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. The thickest band is 50 kDa so our bands should be at about the same height.

Several IMAC and SDS-pages were run, 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 4. Eluent 2 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, since 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.

When the SDS-page had been run we knew that we could produce colicin Fy. The next stage is then to see if it actully works. To be able to prove that our system is working we needed to test it on the actual bacteria, 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 an overnight culture of bacteriocin producing bacteria in 200ml LB 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 [LINK]. The 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 different times. Immediately growth difference could be spotted and after 4 hours the results were very clear as seen in fugure 2.

For more pictures [länk till results]

Sequence and Features