Construction and Characterization of an E. coli bamD Depletion Strain.
Methods Mol Biol
; 1329: 227-43, 2015.
Article
em En
| MEDLINE
| ID: mdl-26427689
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.
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Texto completo:
1
Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Proteínas da Membrana Bacteriana Externa
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Engenharia Genética
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Proteínas de Escherichia coli
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Escherichia coli
Idioma:
En
Revista:
Methods Mol Biol
Ano de publicação:
2015
Tipo de documento:
Article