Difference between revisions of "PMID:10829079"
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| + | !align=left align='left' bgcolor='#CCCCFF' |Citation | ||
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| + | '''Datsenko, KA and 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''':6640-5 | ||
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| + | !align=left align='left' bgcolor='#CCCCFF' |Abstract | ||
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| + | 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. | ||
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| + | !align=left align='left' bgcolor='#CCCCFF' |Links | ||
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| + | [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10829079 PubMed] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC18686 PMC18686] | ||
| + | Online version:[http://dx.doi.org/10.1073/pnas.120163297 10.1073/pnas.120163297] | ||
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| + | !align=left align='left' bgcolor='#CCCCFF' |Keywords | ||
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| + | Chromosomes, Bacterial; DNA Nucleotidyltransferases/metabolism; Escherichia coli/genetics; Integrases; Lac Operon; Mutation; Operon; Plasmids; Polymerase Chain Reaction; Recombinases; Recombination, Genetic | ||
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| + | ==Main Points of the Paper == | ||
| + | {{LitSignificance}} | ||
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| + | == Materials and Methods Used == | ||
| + | {{LitMaterials}} | ||
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| + | ==Phenotype Annotations== | ||
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| + | !|Phenotype of!!Taxon Information!!Genotype Information (if known)!!Condition Information!!OMP ID!!OMP Term Name!!ECO ID!!ECO Term Name!!Notes!!Status | ||
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| + | ==Notes== | ||
| + | |||
| + | ==References== | ||
| + | {{RefHelp}} | ||
| + | <references/> | ||
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| + | [[Category:Publication]] | ||
Latest revision as of 00:08, 20 July 2015
| Citation |
Datsenko, KA and Wanner, BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97:6640-5 |
|---|---|
| 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. |
| Links |
PubMed PMC18686 Online version:10.1073/pnas.120163297 |
| Keywords |
Chromosomes, Bacterial; DNA Nucleotidyltransferases/metabolism; Escherichia coli/genetics; Integrases; Lac Operon; Mutation; Operon; Plasmids; Polymerase Chain Reaction; Recombinases; Recombination, Genetic |
| edit table |
Main Points of the Paper
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Materials and Methods Used
Please list the materials and methods used in this paper (strains, plasmids, antibodies, etc).
Phenotype Annotations
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<protect>
| Phenotype of | Taxon Information | Genotype Information (if known) | Condition Information | OMP ID | OMP Term Name | ECO ID | ECO Term Name | Notes | Status |
|---|---|---|---|---|---|---|---|---|---|
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</protect>
Notes
References
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