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prtN and the pyocin Cluster

In Pseudomonas aeruginosa, the production of R2 type pyocins is regulated by a positive regulator (prtN), alongside a negative one (prtR). The prtN gene activates the expression of pyocin genes by interacting with DNA sequences in noncoding regions of the pyocin cluster. prtR represses the expression of prtN, and therefore acts as a negative regulator of the pyocin cluster.

prtN - BBa_K2943009

In our project, we have separated partN from the genome of Pseudomonas aeruginosa in order to use it as the pyocin cluster activator under our own terms. We have separated prtN from the regulation of prtR, and cloned it in a plasmid under inducible regulation - allowing us to control the expression of prtN, following by the expression of the pyocin cluster.

The pyocin cluster - BBa_K2943000

We have made the composite part BBa_K2943000 - the pyocin cluster, without the tail fiber gene (prf15).

BBa_K2943007 is the first part of the pyocin cluster, containing the promoter of the cluster and genes prf5-prf14. BBa_K2943008 is the second part of the R2 pyocin cluster, containing genes prf16-prf24. Together (BBa_K2943000) we have constructed a cluster that is activated by prtN and is lacking the tail fiber gene. When the cluster is expressed alongside a tail fiber gene (in trans), a pyocin particle will be produced and will target rival bacteria.

Tails

We have made four different tail options for our system. Each tail has a unique targeting spectrum.

BBa_K2943005 is the original tail fiber (with its chaperone) found in the genome of Pseudomonas aeruginosa. This tail fiber targets competing Pseudomonas strains.

BBa_K2943006 is a tail fiber that is made out of a fusion between Pseudomonas aeruginosa tail fiber protein and bacteriophage V10 tail spike protein. Using this part, we are re-targeting R-type pyocins to Escherichia coli O157:H7.

BBa_K2943010 is a fusion between the tail fiber genes of Pseudomonas aeruginosa and bacteriophage P2, alongside a chaperone. Bacteriophage P2, which infects many E. coli strains, has a tail fiber encoded by gene H as well as a chaperone gene, G. We have constructed a fusion between the pyocin's tail fiber and those genes (H and G), resulting with a pyocin that targets a large spectrum of E. coli strains.

BBa_K2943011 is the result of our unique tail finding software! (see software page for more info about our bioinformatic work). We have used our tail finding algorithm and discovered a tail fiber gene from the bacteriophage Fels-2, a tail fiber gene that targets Salmonella Typhimurium and could attach to the pyocin particle. Following this finding, we have build this BioBrick to re-target pyocins against Salmonella Typhimurium.

The composite part BBa_K2943012 consists of two separate BioBrick tails, BBa_K2943006 and BBa_K2943010, each one with its own inducible regulation. This way, under a different sets of molecular inputs, different pyocins with a unique targeting spectrum will be produced.