Difference between revisions of "Part:BBa K1381017"

Latest revision as of 22:00, 17 October 2014

CFY-X6 his

Usage and Biology

CFY, colicin Fy, is a bacteriocin from the colicin family produced by Y. frederiksenii and targets 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 targets 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 causes symptoms[2].

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.

SDS page

To be able to see that colicin Fy was produced we ran an SDS-Page. Before running the SDS-page we extracted the colicin Fy from the inside of the cells by sonication and purification. Since the colicin contains a histidine tag we used IMAC to purify, read protocols [http://2014.igem.org/Team:Uppsala/Project_Notebook here].

Figure 1: 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

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 49,6 kDa. The protein ladder used is Thermo Scientifics PageRuler Unstained Protein Ladder #26614. The thick band in the middle is at 50 kDa, so our colicin should have a band at 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 1. 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.

Inhibition test in Y.enterocolitica
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 [http://2014.igem.org/Team:Uppsala/Project_Notebook protocol]. The supernatant was added to a final concentration of 1%(v/v) in LB with 10^4 CFU/ml Y.enterocolitica. 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 general media, BHI, agar plates at several time steps. After 0 hours growth difference could be spotted and after 4 hours the results were very clear as seen in figure 2.

For more pictures visit results in our [http://2014.igem.org/Team:Uppsala/Project_Killing wiki].

Figure 2. On the left Y. enterocolitica has been grown in liquid culture together with colicin Fy. The right plate is a negative control, where Y. enterocolitica has grown in the same conditions in liquid culture without any colicin Fy added. Both are plated after 4 hours of growth and diluted 1:10.