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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 a rapid and cloning-free gene disruption method (the 3-Step PCR, see Figure) that allows the extension of the lambda-red strategy to many enterobacteriacea such as commensal and pathogenic Escherichia coli, Yersinia, Serratia, Salmonella, Shigella and Klebsiella. This method, which uses linear DNA fragments where homology regions of 500 bp are flanking a selectable resistance marker, improves the efficiency of the lambda-red method for non E. coli K12 bacteria. A full background and protocole is presented in 4 .pdf documents (see here ). .

 

Experimental approach of biofilm formation: continuous-flow microfermentors

The use of an experimental model reproducing all steps of biofilm formation is essential to our biofilm studies. Whereas flow cells, a well established continuous-flow experimental model, allows the non-invasive real-time monitoring of biofilm architecture (Heydorn et al., 2000), they are not adapted for biofilm biomass production . We designed a continuous-flow culture system where biofilm formation can be reproducibly monitored and controlled in micro-fermentors (MF) developed at the Pasteur Institute (see Figure). The biomass recovered from the surface allows further microscopy genetic or biochemistry analysis (i.e. Protein profiling, RNA extraction etc…).

The MF system can be distributed to the academic and industrial community through modest licensing fees (see here ).