Counterselectable marker for Lactobacilli (urgent) - does TetAR work? (Jul/20/2008 )

Hello, I need to delete a gene in Lactobacillus, and wish for a good counterselectable marker.... i was thinking of using TetAR and counterselect with fusaric acid, burt I'm not sure it would work in gram positives, since I haven't been able to find any publications that use it. I've come around some works in which a PRTase such us upp is used, but for what I understood, you need a mutant resitant to a nucleotide analog to begin with, and as my strains are vaccine prototypes, I cannot use any resistant mutants.
Please, help me, I have to finish my thesis project this week and am trapped in this.

Look at the PheS mutant which is non-selective in incorporating p-chlorophenylalanine. It's a single AA substitution in the wt protein and is dominant lethal in the presence of p-chlorophenylalanine. See Peter Kast, plasmid pKSS, 1994. Be sure to adjust the pH of the medium when adding p-chlorophenylalanine (a subtle but very important point). The pheS sequence is almost certainly conserved in the lactobacilli.

thanks a lot, I'll look into it


by the way... does somebody have the pKSS plasmid? I'm not really sure if it will work, as Lactobacilli PheS is not as similar, but it's a start
thanks!