Date of Defense
Leonard Ginsberg, Associate Dean of Arts & Sciences
Silvia Rossbach, Biology
Gyula Ficsor, Biology
The fluorescent substrate, 4-methylumbelliferyl β-D-galactopyranoside can be used in assays for rapid detection of β-galactosidase. The lacZ gene, coding for beta-galactosidase, is an important and commonly used reporter gene. Standardization of flurescent-based detection of the expression of the lacZ gene in Eschericia coli XL1-BLUE containing pBluescript provides a positive control for the development of an efficient method of measurement in genetic toxicology assays measuring expression of DNA reporter genes after exposure to mutagens. The standardization of lacZ expression in E. coli XL1-BLE containing pBluescript KS+ was accomplished by using a standardized enzyme reaction to evaluate enzymatic inhibition in the presence of cell lysing agents. The lysing agents, SDS and chloroform provided the least amount of inhibition. In measuring lacZ gene expression in E. coli XL1-BLUE cells with pBluescript, chloroform lysed the cells better than SDS, resulting in greater fluorescence readings. Salmonella typhimrium TA 1535 containing psk1002 and E. coli XL1-BLUE with pBluescript were exposed to the mutagen, Cisplatin, to determine if the lacZ gene and the lacZ DNA repair reporter gene complex in the Salmonella increase with time in the S. typhimurium TA 1535 containing psk 1002 and remain constant in the E. coli XL1-BLUE with pBluescript.
Meyer, Emily M., "Standardization of Fluorescence-Based Detection of lacZ gene Expression in Eschericia Coli Cells Containing pBluescript" (2001). Honors Theses. 224.
Honors Thesis-Campus Only