Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system.
- Author:
Wei CHEN
1
;
Mei YU
;
Shan-Hu LI
;
Ming-Gang WANG
;
Jian-Guang ZHOU
Author Information
1. Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
- Publication Type:Journal Article
- MeSH:
Bacteriophage lambda;
genetics;
Chromosomes, Bacterial;
genetics;
Escherichia coli;
genetics;
Gene Knock-In Techniques;
methods;
Gene Knockout Techniques;
methods;
Humans;
Lac Operon;
genetics;
Plasmids;
genetics;
Recombination, Genetic
- From:
Chinese Journal of Biotechnology
2005;21(2):192-197
- CountryChina
- Language:Chinese
-
Abstract:
pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.
