Evaluation of three toxin based counterselection systems (pTox) for allelic replacement of Toxin-Antitoxin loci in Variovorax paradoxus EPS
Poster Number
8A
Format
Poster Presentation
Faculty Mentor Name
Paul Orwin
Faculty Mentor Department
Biological Sciences
Graduate Student Mentor Name
Amy Olmos
Abstract/Artist Statement
Toxin-antitoxin (TA) systems are commonly seen in bacterial genomes. Plasmid based TA systems ensure that the plasmid is passed onto daughter cells through post segregational killing. The role of chromosomal encoded TA systems is less clear. The overall project goal is to knock out the TA genes of Variovorax EPS using allelic replacement. The method is based on the insertion of the plasmid into the chromosome by homologous recombination, followed by counterselection to remove the plasmid, leaving behind the deletion construct. The counterselectable toxins are controlled by a rhamnose inducible promoter, and suppressed by glucose in the medium. For this approach to work, the toxins must be effective in the target strain. We compared three pTOX allelic exchange vectors (pTox4, pTox5, pTox6) containing different toxin genes, a purple chromoprotein for verification of insertion, and a selectable antibiotic marker. Appproximately 1kb flanking regions from three TA loci were PCR amplified from the V. paradoxus EPS genome, and cloned into the three vectors using Gibson Assembly to generate the deletion constructs. The plasmids were transformed into E. coli UQ950 and validated by restriction enzyme digestion. These plasmids are mobilized from E.coli to Variovorax paradoxus EPS through conjugation. Transconjugants are grown on media that contains no added amino acids to prevent the growth of the auxotrophic donor strain. Once transconjugants have been obtained, the rhamnose induced toxin is used to select against the inserted plasmid. Recombinants that have lost the toxin, the purple chromoprotein, and the antibiotic marker will then be tested for allelic replacement by PCR.
Location
Information Commons, William Knox Holt Memorial Library and Learning Center
Start Date
29-4-2023 10:00 AM
End Date
29-4-2023 1:00 PM
Evaluation of three toxin based counterselection systems (pTox) for allelic replacement of Toxin-Antitoxin loci in Variovorax paradoxus EPS
Information Commons, William Knox Holt Memorial Library and Learning Center
Toxin-antitoxin (TA) systems are commonly seen in bacterial genomes. Plasmid based TA systems ensure that the plasmid is passed onto daughter cells through post segregational killing. The role of chromosomal encoded TA systems is less clear. The overall project goal is to knock out the TA genes of Variovorax EPS using allelic replacement. The method is based on the insertion of the plasmid into the chromosome by homologous recombination, followed by counterselection to remove the plasmid, leaving behind the deletion construct. The counterselectable toxins are controlled by a rhamnose inducible promoter, and suppressed by glucose in the medium. For this approach to work, the toxins must be effective in the target strain. We compared three pTOX allelic exchange vectors (pTox4, pTox5, pTox6) containing different toxin genes, a purple chromoprotein for verification of insertion, and a selectable antibiotic marker. Appproximately 1kb flanking regions from three TA loci were PCR amplified from the V. paradoxus EPS genome, and cloned into the three vectors using Gibson Assembly to generate the deletion constructs. The plasmids were transformed into E. coli UQ950 and validated by restriction enzyme digestion. These plasmids are mobilized from E.coli to Variovorax paradoxus EPS through conjugation. Transconjugants are grown on media that contains no added amino acids to prevent the growth of the auxotrophic donor strain. Once transconjugants have been obtained, the rhamnose induced toxin is used to select against the inserted plasmid. Recombinants that have lost the toxin, the purple chromoprotein, and the antibiotic marker will then be tested for allelic replacement by PCR.