Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli

doi:10.1016/0378-1119(91)90366-J

Gene

Volume 100, April 1991, Pages 195-199

https://doi.org/10.1016/0378-1119(91)90366-J Get rights and content

Abstract

Using the polymerase chain reaction and standard recombinant DNA techniques, a series of new multipurpose low-copy-number (Icn) vectors, pWSK29, pWKS30, pWSK129 and pWKS130, have been constructed. Plasmids pWSK29 and pWKS30 carry the ampicillin-resistance marker (Ap^R), 20 unique cloning sites flanked by T7 and T3 RNA polymerase promoters, the lacZα. gene and the bacteriophage fl origin of replication (ori) for production of single-stranded (ss) DNA in the presence of a helper phage. Plasmids pWSK129 and pWKS130 carry the kanamycin-resistance marker (Km^R) and have 16 unique cloning sites flanked by T7 and T3 RNA polymerase promoters positioned within the lacZα gene. Plasmids pWSK129 and pWKS130 also contain the fl ori for the generation of ss DNA in the presence of a helper phage. All of the plasmids have an lcn of six to eight per cell. Each vector can be used for: (i) complementation analysis, (ii) generating unidirectional deletions with exonuclease III/S1 nuclease, (iii) DNA sequencing, (iv) high-level gene expression using T7 RNA polymerase, and (v) run-off transcription. They are very useful for analyzing genes encoding proteins which are toxic in Escherichia coli in high copy number.

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Gene

Abstract

References (20)

Unidirectional digestion with exonuclease III create targets breakpoints for DNA sequencing

Gene

Direct sequencing of denatured plasmid DNA

Methods Enzymol.

Versatile low-copy-number plasmid vectors for cloning in Escherichia coli

Gene

The pUC plasmids, an M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers

Gene

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Gene

Nucleotide sequence of the gene ompA encoding the outer membrane protein II of Escherichia coli K-12

Nucleic Acids Res.

Complete sequence of pSC101

Nucleic Acids Res.

XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection

Bio/Technology

Cloning of the altered mRNA stability (ams) gene of Escherichia coli K-12

J. Bacteriol.

Construction of biologically functional bacterial plasmids in vitro

Cited by (1037)

Anaerobic respiration of host-derived methionine sulfoxide protects intracellular Salmonella from the phagocyte NADPH oxidase

High fat intake sustains sorbitol intolerance after antibiotic-mediated Clostridia depletion from the gut microbiota

Persistent Salmonella infections in humans are associated with mutations in the BarA/SirA regulatory pathway

Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection

Human variation impacting MCOLN2 restricts Salmonella Typhi replication by magnesium deprivation

Transcriptional insight into the effect of benzalkonium chloride on resistance and virulence potential in Salmonella Typhimurium