PMID- 10829079
OWN - NLM
STAT- MEDLINE
DCOM- 20000713
LR  - 20240109
IS  - 0027-8424 (Print)
IS  - 1091-6490 (Electronic)
IS  - 0027-8424 (Linking)
VI  - 97
IP  - 12
DP  - 2000 Jun 6
TI  - One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR 
      products.
PG  - 6640-5
AB  - 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.
FAU - Datsenko, K A
AU  - Datsenko KA
AD  - Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, 
      USA.
FAU - Wanner, B L
AU  - Wanner BL
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - Proc Natl Acad Sci U S A
JT  - Proceedings of the National Academy of Sciences of the United States of America
JID - 7505876
RN  - 0 (Recombinases)
RN  - EC 2.7.7.- (DNA Nucleotidyltransferases)
RN  - EC 2.7.7.- (Integrases)
RN  - EC 2.7.7.- (integron integrase IntI1)
SB  - IM
MH  - *Chromosomes, Bacterial
MH  - DNA Nucleotidyltransferases/metabolism
MH  - Escherichia coli/*genetics
MH  - *Integrases
MH  - Lac Operon
MH  - *Mutation
MH  - Operon
MH  - Plasmids
MH  - *Polymerase Chain Reaction
MH  - Recombinases
MH  - Recombination, Genetic
PMC - PMC18686
EDAT- 2000/06/01 09:00
MHDA- 2000/07/15 11:00
PMCR- 2000/12/06
CRDT- 2000/06/01 09:00
PHST- 2000/06/01 09:00 [pubmed]
PHST- 2000/07/15 11:00 [medline]
PHST- 2000/06/01 09:00 [entrez]
PHST- 2000/12/06 00:00 [pmc-release]
AID - 120163297 [pii]
AID - 1632 [pii]
AID - 10.1073/pnas.120163297 [doi]
PST - ppublish
SO  - Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. doi: 10.1073/pnas.120163297.