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.

3- Primers in the 3S-PCR: design and use

4- 3S-PCR: a step-by-step bench protocol

For 3S-PCR applications, see:

Escherichia coli
• Beloin, C et al. (2004) Global impact of mature biofilm lifestyle on E. coli K12 gene expression. Mol Microbiol. 51 (3), 659–674
• A. Roux et al. (2005) Combined Inactivation and Expression Strategy To Study Gene Function under Physiological Conditions: Application to Identification of New Escherichia coli Adhesins J Bacteriol.187:1001-13.
Yersinia sp
• Derbise, A et al. (2003)« A rapid and simple method for inactivating chromosomal genes in Yersinia".. FEMS Immunol Med Microbiol. Sep 22;38(2):113-6.
Salmonella enteritidis
• Solano, C.,et al. (2002). "Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose." Mol Microbiol 43(3): 793-808.
Serrratia marcescens
• Rossi, M.S. et al. . (2003) « .Hemophore-mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules". Mol Microbiol. 48 (6) :1467–1480.
Shigella flexneri
• Beloin, C., et al. (2003) Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein. Mol Genet Genomics 270: 66-77.