Patent ID: 8628967
Filing Date: 2014-01-14
Classification: C12N

Abstract:
1. A process for the replacement of a target DNA sequence by homologous recombination in Clostridia, wherein the process comprises the following steps: transforming a strain with a vector comprising: an origin of replication permitting its replication in Clostridia, and a replacement cassette comprising a first marker gene surrounded by two sequences homologous to selected regions around the target DNA sequence, allowing the recombination of the cassette, and a second marker gene, selecting strains having integrated in their genome said cassette that express the first marker gene, and selecting strains having eliminated said vector that do not express the second marker gene, wherein the second marker gene is a counter-selectable marker that restores the activity of a non-essential absent or deleted gene and whose presence is lethal in the presence of its cognate substrate, chosen among the list consisting of: genes giving sensitivity to 5-fluoro-uracile (5-FU), genes giving sensitivity to gamma-glutamyl hydrazide (GBS), genes giving sensitivity to 8-aza-2,6-diaminopurine (8ADP), genes giving sensitivity to sucrose, genes giving sensitivity to streptomycin, genes giving sensitivity to fusaric acid, pheS gene, thyA gene, lacY gene, gata-I gene, and ccdB gene.