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Plasmid

About: Plasmid is a(n) research topic. Over the lifetime, 44320 publication(s) have been published within this topic receiving 1997391 citation(s). The topic is also known as: plasmids.
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Journal ArticleDOI
01 Jan 1985-Gene
TL;DR: New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors and mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences.
Abstract: Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.

14,826 citations


Journal ArticleDOI
Abstract: A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

13,689 citations


Journal ArticleDOI
TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.
Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

13,178 citations


01 Jan 1979-
TL;DR: The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes, and achievesequate pH control without using a pH meter.
Abstract: Aprocedure forextracting plasmid DNAfrombacterial cellsis described. Themethodissimpleenough topermit theanalysis bygel electrophoresis of100ormoreclonesperdayyetyieldsplasmid DNAwhichis pureenough tobedigestible byrestriction enzymes.Theprinciple ofthe methodisselective alkaline denaturation ofhighmolecular weightchromosomal DNAwhilecovalently closedcircular DNAremains double-stranded. Adequate pHcontrol isaccomplished without usinga pHmeter.Uponneutralization, chromosomal DNArenatures toformaninsoluble clot,leaving plasmid DNAin thesupernatant. Largeandsmallplasmid DNAshavebeenextracted bythis method.

13,158 citations


Journal ArticleDOI
TL;DR: Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmid molecules.
Abstract: Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.

10,867 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202213
2021802
2020896
2019962
2018906
2017896

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Topic's top 5 most impactful authors

Donald R. Helinski

113 papers, 14.5K citations

Stanley N. Cohen

90 papers, 16.3K citations

Alfred Pühler

79 papers, 15.2K citations

Christopher M. Thomas

64 papers, 5.2K citations

Alessandra Carattoli

46 papers, 7.8K citations