title: Construction and Characterization of an E. coli bamD Depletion Strain.
abstract: The central Bam components BamA and BamD are both essential genes in E. coli, a fact that often confounds genetic analysis using classical methods. The isolation of "depletion strains" in which these genes can be conditionally expressed removes this obstacle and facilitates the in vivo characterization of Bam function. This chapter describes an efficient two-step recombineering method for the construction of such a depletion strain, which contains an arabinose-inducible allele of bamD, using the λ Red system. Additionally, a simple protocol is presented for the depletion of bamD expression in live cells, which is particularly useful for the characterization of mutant alleles of bamD (complementation analysis). In principle, the procedures described can be adapted to produce and characterize depletion strains for any essential gene in E. coli or any other bacterium that is similarly amenable to genome engineering.
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, 279 Campus Drive West, Beckman Center, Stanford, CA, 94305, USA. danter@stanford.edu.
authors.forename: Dante P
authors.initials: DP
authors.lastname: Ricci
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D011995:Recombination, Genetic
chemical_list: D001425:Bacterial Outer Membrane Proteins; C513739:BamD protein, E coli; D029968:Escherichia coli Proteins
keywords: BAM complex; BamA; BamD; Beta-barrel membrane proteins; Complementation analysis; E. coli