[Site-directed mutagenesis and promoter functional analysis of RM07 DNA fragment from Halobacterium halobium in Escherichia coli].
The RM07 DNA fragment from Halobacterium halobium was inserted upstream of chloramphenicol acetyltransferase (cat) reporter gene in pKK232-8 in two different orientations (positively or negatively), generating the RM07-cat fusion plasmids pRM07-1 (+) and pRM07-1 (-). These two plasmids and pKK232-8 were transformed into Escherichia coli HB101 respectively, then the antibiotic resistance level and the chloramphenicol acetyltransferase protein concentration of different transformants were detected. The research results revealed that HB101/pRM07-1 (+) was resistant to chloramphenicol and could grow on the plate containing chloramphenicol, but HB101/pRM07-1 (-) and HB101/pKK232-8 were sensitive to it. Therefore, the results suggested that the positive RM07 fragment had promoter activity in Escherichia coli while the negative RM07 fragment did not. Site-directed mutagenesis of RM07 was performed by PCR mutagenesis method. The effect of specific nucleotide mutations on the chloramphenicol resistance level of different transformants was detected. The different transformants containing different nucleotide mutations were inoculated on the plates containing various concentrations of chloramphenicol and then incubated at the same time. The change of chloramphenicol resistance level reflected the change of promoter activity. By using this method, the important nucleotide responsible for the promoter function of RM07 in Escherichia coli was determined. The promoter activity of RM07 in Escherichia coli was also improved greatly by modifying the nucleotide component.
Yang
Yang
Y
Department of Biotechnology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Ping
Shen
S
D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004269:DNA, Bacterial / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics*; D019617:Halobacterium salinarum / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology*; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D015500:Chloramphenicol O-Acetyltransferase