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Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli
Li, Hedan; Hao, Chengwei & Xu, Daqing
Abstract
Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However,
there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic
and applied research on extremely toxic proteins.
Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the
efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator
upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in
addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the
antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and
L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly
repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of
the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter
efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA,
ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of
expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing
the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli
JM109(DE3).
Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.
Keywords
pAU10 vector; T7-lacO hybrid promoter; rrnBT2 terminator; Leaky transcription; trp promoter/operator; Antisense RNA; Tight regulation; IPTG inducer; L-tryptophan corepressor; Efficient expression
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