Construction and characterization of a biotin-regulated gene expression system in Escherichia coli.
An autoregulated gene expression system in Escherichia coli was designed such that the cloned genes on the vector were not expressed until biotin was depleted during cell growth. The expression vectors were constructed by assembling the DNA fragments containing the regulatory region of the E. coli biotin operon (bio operon), the universal ribosome-binding site (RBS) and the strong transcription terminator rrnBT1T2. The promoter region was further modified by site-directed mutagenesis to create promoters of varied strength. The feasibility of this system was examined in E. coli strain R901 (with bio operon deleted) using various marker genes, including the E. coli birA gene, T7 RNA polymerase gene and yellowfin-porgy growth-hormone gene. The results demonstrated that the induction of marker-gene expression can be triggered as the biotin concentration drops to a threshold value of approximately 2 ng/mL by metabolic utilization.
Chang
Y S
YS
Department of Biology, National Sun Yat-Sen University, Kaohsiung, Taiwan, ROC.
Shiuan
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D001710:Biotin / Q000502:physiology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005240:Feasibility Studies; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005822:Genetic Vectors; D006706:Homeostasis / Q000502:physiology; D009876:Operon; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000502:physiology
D012097:Repressor Proteins; D001710:Biotin