Construction and application of the vectors to identify genes encoding exported proteins of Escherichia coli.
In order to clone genes having signal sequences of Escherichia coli, four vectors with or without Lac or Ara promoter were constructed using a leaderless β-lactamase as reporter. Fragments of tetracycline resistance gene (Tet) with or without promoter were used to confirm the vectors' ability to clone and report signal sequences. The minimum inhibitory concentration of ampicillin of the transformants was measured to detect the expression and secretion efficiency of the vectors. The results showed that the β-lactamase could be co-expressed and secreted with Tet protein. The Lac or Ara promoter in the vectors could be regulated by different inducers, and the Ara promoter showed higher regulative efficiency than the Lac. The best induction dose of L-arabinose for the Ara promoter is 1.25 %. All the four vectors were stably maintained in host after being inoculated for 20 passages in antibiotics-free media. Genomic library of an avian pathogenic strain, E. coli O2, was constructed using the pMB-Ara-T vector we developed. 318 clones were obtained from the genomic library of E. coli strain O2, and the inserts in these clones represented 276 genes based on sequence analysis. Among the 276 cloned fragments, only 128 had complete promoter sequence. For the 128 fragments with promoter, only 27 could be expressed under LB culture condition without inducer, the other 101 were only expressed under induction. The results showed our constructed vectors could efficiently capture all kinds of exported protein genes in vitro, including the ones without promoter or with inactive promoter.
College of Animal Sciences, Zhejiang University, Hangzhou, 310058, China.
Dong
D
Niu
Qinfang
Q
Shen
Junli
J
Zhu
Jiangmei
J
Liu
Jiajie
J
Yuan
Shuang
S
Tan
Xuping
X
Yu
D000667:Ampicillin / Q000494:pharmacology; D001089:Arabinose / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000378:metabolism; D015203:Reproducibility of Results; D014170:Transformation, Genetic / Q000187:drug effects
D029968:Escherichia coli Proteins; D000667:Ampicillin; D001089:Arabinose