3-step PCR (3S-PCR) protocol for lambda red gene inactivation in Gram-negative bacteria using pKOBEG or other linear DNA recombinogenic plasmids
In collaboration with E. Carniel's group, (Yersinia Unit, Institut Pasteur), we developped, in parallel with other laboratories, a rapid PCR-based method to inactivate target genes in E. coli. These methods, most of them originally derived from K.C Murphy's observations, work well for Escherichia coli and have been described independently by several groups (see references within the enlarged figure on the right). They are based on the use of very short regions homologous to the target genes in 2 long primers used to amplify a selective marker. The linear PCR fragment is then introduced in competent cells transiently expressing the lambda red (or RecET) functions (see Figure).
We developped a cloning-free disruption method (the 3-Step PCR, see below), which uses linear DNA fragments where homology regions of 500 bp are flanking a selectable resistance marker. This method, extends the efficiency of the lambda-red method to many enterobacteriacea.