Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli

2008 ◽  
Vol 373 (2) ◽  
pp. 389-391 ◽  
Author(s):  
Jing Li ◽  
Chunhua Li ◽  
Wei Xiao ◽  
Dongxia Yuan ◽  
Gang Wan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Plasmid Template ◽  
Dpni Digestion

An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

2012 ◽  
Vol 195-196 ◽  
pp. 407-411
Author(s):  
Mu Qing Qiu
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

scholarly journals Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase

1992 ◽  
Vol 267 (32) ◽  
pp. 22830-22836 ◽  
Author(s):  
K Ostanin ◽  
E.H. Harms ◽  
P.E. Stevis ◽  
R Kuciel ◽  
M.M. Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Phosphatase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

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