Article ID Journal Published Year Pages File Type
1176999 Analytical Biochemistry 2010 8 Pages PDF
Abstract

In genetic engineering, gene expression is often modulated by replacements in promoter regions. Any deliberate intervention into the regulatory elements requires a subsequent evaluation based on analysis of reporter proteins. We have developed a new and rapid approach for characterization of promoter activity in which promoter strengths are determined by antibiotic resistance level. Values are expressed in comparison with those obtained from the reference promoter using the kanamycin resistance (aminoglycoside 3′-phosphotransferase) gene as a reporter. The new assay vector pSB1K0prom enables straightforward cloning of promoters or their subparts; therefore, mutations in different elements of the promoter region are easily introduced and analyzed. A series of promoters can be examined in parallel because no protein analysis is required other than determination of bacterial growth rates in the presence of increasing kanamycin concentrations. An internet application called PromCal for evaluation of experimental data has also been developed and is freely accessible at http://web.fkkt.uni-lj.si/biokemija/nskrlj/tools/PromCal.php.

Related Topics
Physical Sciences and Engineering Chemistry Analytical Chemistry
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