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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Edgar RS (1966) Conditional lethals. In Phage and the Origin of Molecular Biology, edited by J. Cairns, G. S. Stent and J. B. Watson. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp. 166–170
Wu T, Malinverni J, Ruiz N et al (2005) Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli. Cell 121:235–245
Malinverni JC, Werner J, Kim S et al (2006) YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli. Mol Microbiol 61:151–164
Misra R, Stikeleather R, Gabriele R (2014) In vivo roles of BamA, BamB and BamD in the biogenesis of BamA, a core protein of the β-barrel assembly machine of Escherichia coli. J Mol Biol. (in press)
Rossiter AE, Leyton DL, Tveen-Jensen K et al (2011) The essential BAM complex components BamD and BamA are required for autotransporter biogenesis. J Bacteriol 193(16):4250–4253
Kim S, Malinverni JC, Sliz P et al (2007) Structure and function of an essential component of the outer membrane protein assembly machine. Science 317:961–964
Boyd D, Weiss DS, Chen JC et al (2000) Towards single-copy gene expression systems making gene cloning physiologically relevant: lambda InCh, a simple Escherichia coli plasmid-chromosome shuttle system. J Bacteriol 182:842–847
Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388
Yu D, Ellis HM, Lee EC et al (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA 97:5978–5983
Thomason L, Court DL, Bubunenko M, et al. (2007) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol Chapter 1:Unit 1.16 (Editors: Frederick M, Ausubel Al)
Guzman LM, Belin D, Carson MJ et al (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
Schleif R (2010) AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev 34:779–796
Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res 38:e92
Sheppard DE, Englesberg E (1967) Further evidence for positive control of the L-arabinose system by gene araC. J Mol Biol 25:443–454
Wilcox G (1974) The interaction of L-arabinose and D-fucose with AraC protein. J Biol Chem 249:6892–6894
Brockmann B, Koop Genannt Hoppmann KD, Strahl H et al (2013) Time-delayed in vivo assembly of subunit a into preformed Escherichia coli FoF1 ATP synthase. J Bacteriol 195:4074–4084
Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
Horton RM, Hunt HD, Ho SN et al (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
Shetty RP, Endy D, Knight TF (2008) Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2:5
Quan J, Tian J (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4:e6441
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645
Rhodius VA, Suh WC, Nonaka G et al (2006) Conserved and variable functions of the sigmaE stress response in related genomes. PLoS Biol 4:e2
Bury-Moné S, Nomane Y, Reymond N et al (2009) Global analysis of extracytoplasmic stress signaling in Escherichia coli. PloS Genet 5:e1000651
Acknowledgements
I wish to express my indebtedness to Marcelo Sousa (University of Colorado Boulder) for guidance and insightful comments on this manuscript, and to Tom Silhavy (Princeton University) and Natacha Ruiz (Ohio State University) for support and encouragement.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ricci, D.P. (2015). Construction and Characterization of an E. coli bamD Depletion Strain. In: Buchanan, S., Noinaj, N. (eds) The BAM Complex. Methods in Molecular Biology, vol 1329. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2871-2_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2871-2_18
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2870-5
Online ISBN: 978-1-4939-2871-2
eBook Packages: Springer Protocols