Difference between revisions of "Part:BBa K2943005"

Latest revision as of 23:02, 21 October 2019

prf15 and prf16 from Pseudomonas aeruginosa PAO1

The tail fiber protein (Prf15) and its chaperone (Prf16) allows Pseudomonas aeruginosa's pyocin complexes 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 and perforating rival bacteria. In other words, the tail fiber protein determines the target bacteria’s identity.

The Assembly of the prf15 and prf16 expression plasmid

Following earlier researches, we concluded that the both the tail fiber and its chaperone should exist in the same plasmid for optimal expression [1]. We designed and ordered an adequate G-Block containing both of prf15 and prf16 and inserted it into a the backbone of the je278 plasmid (Received from the Elbaz lab). This plasmid allows induction by iPTG which we later used for expressing our proteins. The plasmid and the gBlock were assembled via the Gibson assembly method and later transformed into E.coli BL21 bacteria. Later on, we used a miniprep kit to extract and sequence the plasmid, in order to validate that the Gibson assembly was assembled correctly and that no mutations appeared.

Protein expression

Finally, once the plasmid was sequenced, we went to check our protein expression. We used iPTG and preformed protein gel expression test to make sure both proteins express as expected [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.

Demonstrating the wild-type potential

In previous researches [1,2 and 3] it has been shown that expression plasmids such as the one we constructed can generate a functioning pyocin when combined with another plasmid containing the rest of the pyocin cluster, even if the two are separated. Following these researches we set out to preform a plaque assay demonstrating our system. The first step was preforming this plaque assay with the wild type Pseudomonas aeruginosa PAO1: Using our plaque assay protocol [4], 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. We then pipetted some of 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] 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.

Our future plans included preforming the same protocol, this time with the engineered BBa_K2943005 combined with another BioBrick containing the rest of the pyocin cluster genes, to create engineered pyocins. However we had challenges with expressing the rest of the complex and thus, we have not achieved this. we showed that the pyocin mechanism of Prf15 and Prf16was validated in several ways. first, it has been researched before, second we've managed to clone it into a plasmid and then express the protein as expected (as can be seen in Fig. 2). Even tough we did not fully implement our original plan, The plaque assay we preformed with the WT system, demonstrates the full potential of the concept and supports our work.

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.
[4] Plaque Assay Protocol - https://2019.igem.org/wiki/images/a/a8/T--TAU_Israel--Protocols-cloning.pdf
[5] iGEM's TAU tail plasmid assembly notebook - https://2019.igem.org/wiki/images/4/48/T--TAU_Israel--Protocols-tails_plasmids_cloning_notebook.pdf

Sequence and Features