[Function of promoter DNA fragments from halophilic archaea in Escherichia coli].
RM07 and RM13 DNA fragments could function as promoter in Escherichia coli, which were isolated from an archaeon Halobacterium halobium R1. In the present study, promoter activities of these two fragments were confirmed by beta-galactosidase activity analysis and microcalorimetric studies. They were cloned into promoter-probe vector pYLZ-2 respectively. Four recombinant strains TE07, TE07-2, TE131 and TE132 were obtained, and all fragments were found to be active in E. coli DH5alpha. The beta-galactosidase activity of TE132 was higher than that of TE07-2. Both TE07 and TE131 had weak beta-galactosidase activity. Then the heat output of E. coli DH5alpha and its transformants had been detected by a microcalorimetric method at 37 degrees C. Compared with E. coli DH5alpha, the growth rate constant of E. coli T2 (pYLZ-2), TE07, TE07-2, TE131 and TE132 strain was reduced 6.5%, 11%, 41.1%, 47.5% and 42.7% respectively. When IPTG was added to LB medium, beta-galactosidase activity and heat output had been enhanced slightly in all strains. The results suggested that there was close correspondence between promoter activity and microcalorimetric results, and the heat output of growth was mainly affected by gene expression in E. coli. The higher beta-galactosidase activity of E. coli was, the lower its growth rate constant was. At the meantime, Microcalorimetric studies implied that 700bps of RM13 ( RM131) fragment would have stronger promoter activity than RM13. Microcalorimetry may be used as a new approach for analyzing the regulation of foreign gene expression.
Huang
Yu-Ping
YP
College of Life Science, Wuhan University, Wuhan 430072, China. yphuang86@yahoo.com.cn
Liu
Peng
P
Liu
Yi
Y
Shen
Yun-Fen
YF
Shen
Ping
P
D019641:DNA, Archaeal / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017930:Genes, Reporter; D019617:Halobacterium salinarum / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D019641:DNA, Archaeal