Difference between revisions of "Part:BBa K2943005"

Revision as of 13:48, 21 October 2019

prf15 and prf16 from Pseudomonas aeruginosa PAO1

Biology: Tail fiber protein (Prf15) and its' chaperone (Prf16) allows Pseudomonas aeruginosa's Pyocin particles to target rival Pseudomonas strains. They act as a homo-trimer that forms a tail which connects to the base-plate of the Pyocin on one side, and to the membrane of the rival bacteria on the other. This attachment is the first step in the process of attacking rival bacteria. The tail fiber determines the specific target that can be destroyed and will be only expressed after an SOS response, such as DNA damage.

We have validated the mechanism of Prf15 and Prf16 as part of our wet lab work in 2 ways:
1. We created a plasmid which contains prf15 and prf16:
We inserted a gblock of prf15 and prf16 into the backbone vector using Gibson Assembly.
The backbone name is je278, has ampicillin resistance and a promoter under IPTG regulation. We transformed the Gibson results into E.coli BL21 bacteria. After that we used miniprep kit to extract and sequence the plasmid and confirmed the Gibson was successful without mutation.
In addition, we used IPTG to induce Prf15 and Prf16, and used protein gel to make sure they were expressed [Fig 1]. For control we used bacteria that were not induced by IPTG.


[Fig 1]Left: Protein Gel for induced prf15 and prf16.
Right: Transformation plates for the Gibson results. Right plate shows the self reaction results and left plate shows prf15- prf16 plasmid results.

2. We have done a plaque assay with the wild type Pseudomonas aeruginosa PAO1:
Using the plaque assay protocol that can be found here: https://2019.igem.org/wiki/images/a/a8/T--TAU_Israel--Protocols-cloning.pdf, we tested the pyocin system in the WT PAO1. After inducing SOS response in the bacteria with Mitomycin C, we extracted the lysate that contains the pyocin. Then, we pipette the lysate down on a plate that contained the target strains. As control, we used PAO1 that did not receive Mitomycin C and therefore did not undergo SOS response and created pyocin. No target bacteria grew on the area where we pipette the WT's lysate.[Fig 2]


[Fig 2] Plaque assay results. White circles marks area with no growth of bacteria.

In conclusion: we showed that the pyocin mechanism of Prf15 and Prf16 is working in several ways. The existence of the Prf15 and Prf16 in the protein gel, added with the success of the plaque assay experiment, have demonstrated the potential of the pyocin system to kill bacteria.

Link to the our tail plasmid assembly notebook: https://2019.igem.org/wiki/images/4/48/T--TAU_Israel--Protocols-tails_plasmids_cloning_notebook.pdf
Reference:
[1] Williams, Steven R., et al. "Retargeting R-type pyocins to generate novel bactericidal protein complexes." Appl. Environ. Microbiol. 74.12 (2008): 3868-3876.
[2]Scholl, Dean, et al. "An engineered R-type pyocin is a highly specific and sensitive bactericidal agent for the food-borne pathogen Escherichia coli O157: H7." Antimicrobial agents and chemotherapy 53.7 (2009): 3074-3080.
[3] Scholl, Dean M., and Steven R. Williams. "Modified bacteriocins and methods for their use." U.S. Patent No. 7,700,729. 20 Apr. 2010.
Sequence and Features