A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli.
Temperature-sensitive (TS) mutants of a gene are ones of which the activity or phenotype is very similar to that of wild type only at certain temperature and they provide extremely powerful tool for studying protein function in vivo. Here we report a novel strategy to generate TS phenotype of the interest gene in Escherichia coli based on a temperature-sensitive T7-expression system. A TS T7-RNA polymerase is generated by interrupting it with a TS intein from Saccharomyces cerevisiae vacuolar ATPase subunit (VMA), resulting that the gene flanked by T7-promoter and T7-terminator will be transcribed only at the permissive temperature (18 degrees C), not at the restrictive temperature (37 degrees C). The feasibility to create TS phenotype of this strategy was detected using lacZ as target. Reverse transcriptase polymerase chain reaction (PCR) indicated that at 18 degrees C, transcripts of T7-promoter controlled lacZ were at least 85 times more than those at 37 degrees C. Western blot analysis and enzymatic assay showed that large amounts of active His6-tagged LacZ produced at 18 degrees C but little at 37 degrees C. This strategy appears more promising than other TS creation methods because the target is pre-designed, no modification is introduced, and only simple DNA manipulation is required.
Liang
Rubing
R
College of Life Science and Technology, Shanghai Jiaotong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Liu
Xipeng
X
Liu
Jianhua
J
Ren
Qiushi
Q
Liang
Peiji
P
Lin
Zhixin
Z
Xie
Xiangming
X
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D047668:Inteins / Q000235:genetics; D007763:Lac Operon / Q000235:genetics / Q000502:physiology*; D008828:Microbiological Techniques; D009154:Mutation; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases