Bypass trapped flow analysis system (ByT-FAS) used in application: quantitative chemiluminescent detection of whole intact E. coli cell genetic transcription levels via induction of luciferase with tetracycline.
Bypass trapped flow analysis system (ByT-FAS) is a new trapped flow apparatus designed to perform chemical measurements with flow injection systems at physical steady state using small volumes (10-150 mul) of injected sample and reagent. We have used a micro-volume version of ByT-FAS instrumentation with a chemiluminescent detection system to quantitate the protein transcription levels of transformed whole intact E. coli cells. The cells were transformed using a firefly luciferase encoding plasmid with a tetracycline inducible promoter. Luciferase synthesis was induced in E. coli cells containing multiple copies of this plasmid by different levels of tetracycline in the growth media. The level of induction was determined by measuring the velocity of luciferase enzyme per absorbance unit of the injected culture. The micro-volume ByT-FAS instrumentation permitted the rapid determination of the level of induced luciferase and was significantly faster than the traditional quantitative determination of genetic transcription levels. The micro-volume ByT-FAS assays also used significantly lower amounts of the expensive substrate luciferin. This is the first reported use of ByT-FAS for the detection and analyses of transformed cells. ByT-FAS with chemiluminescent detection has the potential of being a useful tool for the rapid analyses of promoter DNA sequences, promoters, and transcription repressors in whole intact bacterial cells by molecular biologists and biochemists.
Hillard
S W
SW
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, 304 Engel Hall, Blacksburg, Virginia 24 060, USA.
Stewart
K K
KK