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Construction and characterization of a biotin-regulated gene expression system inEscherichia coli

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Abstract

An autoregulated gene expression system inEscherichia 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 theE. 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 inE. coli strain R901 (withbio operon deleted) using various marker genes, including theE. 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.

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Abbreviations

bio operon:

biotin operon

bp:

base pair(s)

kb:

kilo base-pair(s)

nt:

nucleotide(s)

o:

operator

RBS:

ribosome-binding site

T-broth:

trypone broth

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Authors and Affiliations

  1. Department of Biology and Institute of Life Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan, ROC

    Yuo-Sheng Chang & David Shiuan

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  1. Yuo-Sheng Chang
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  2. David Shiuan
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Chang, YS., Shiuan, D. Construction and characterization of a biotin-regulated gene expression system inEscherichia coli . Appl Biochem Biotechnol 66, 147–158 (1997). https://doi.org/10.1007/BF02788759

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