Expression of green fluorescent protein in Bacillus brevis under the control of a novel constitutive promoter F1 and insertion mutagenesis of F1 in Escherichia coli DH5alpha.
The constitutive expression vector pHY300-F1gfp was constructed to test the function of a promoter, F1, cloned from the rice epiphyte Bacillus brevis strain DX01. The DX01 cells harboring the plasmid pHY300-F1gfp were shown to produce bright green fluorescence. The results were confirmed by Western blot analysis and fluorescence-activated cell sorting. Expression of the F1 promoter was constitutive. To improve the activity of F1, insertion mutagenesis of F1 based on in vitro transposition reaction was performed. Seven mutants with enhanced transcription activity in Escherichia coli DH5alpha were obtained. The enhanced promoters showed similar high activities in B. brevis strain DX01.
Chen
Yunpeng
Y
Institute of Genetics, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, 220 Handan Rd, Shanghai 200433, PR China.
Yan
Jian
J
Yang
Mingjie
M
Wang
Jingwen
J
Shen
Daleng
D
D001407:Bacillus / Q000235:genetics*; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic / Q000235:genetics*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins