[Construction of recombinant plasmid using Neo/E Technology].
A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.
Li
Shan-Hu
SH
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
Wang
Jian
J
Li
Jie-Zhi
JZ
Huang
Cui-Fen
CF
Zhou
Jian-Guang
JG
D010582:Bacteriophage lambda / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D010957:Plasmids / Q000235:genetics*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D011938:Rec A Recombinases