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Evaluation of the tetracycline promoter system for regulated gene expression in Kluyveromyces marxianus
Author(s) -
Pecota Douglas C.,
Da Silva Nancy A.
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20584
Subject(s) - tetr , repressor , biology , microbiology and biotechnology , promoter , gene , derepression , activator (genetics) , inducer , ura3 , gene expression , aurintricarboxylic acid , reporter gene , plasmid , biochemistry , psychological repression , apoptosis , programmed cell death
A tetracycline repressible promoter system designed for Saccharomyces cerevisiae was evaluated for use in Kluyveromyces marxianus . A plasmid was constructed containing the Escherichia coli β‐glucuronidase ( gus ) gene cloned downstream of the yeast tet‐off promoter, the tetR‐VP16 activator protein gene, and the URA3 gene for selection. The tet‐off promoter‐ gus construct was integrated into the chromosomal DNA and tested under varying growth conditions in complex medium. The repressors tetracycline and doxycycline were both found to be effective for inhibiting gene expression. Doxycycline levels of 0.5 µg/mL or greater were sufficient to nearly completely suppress Gus synthesis. For most transformants, the induction ratio was approximately 2,000‐fold. The tet‐off promoter was effective at 30, 37, and 42°C, although the overall Gus activity was highest at 37°C. During exponential growth, little product was formed; expression increased dramatically in late exponential and early stationary phase. The promoter thus shows promise for protein synthesis following cell growth. No inducer is required and the repressor is only needed to prevent expression during the seed culture. © 2005 Wiley Periodicals, Inc.