Nuclear Transformation of Chlamydomonas Reinhardtii to Allow for Studies on Circadian Expression of the β-Tubulin Gene

Circadian rhythms are rhythms that are capable of free-running in constant conditions with a period of approximately 24 hours, that are able to compensate temperature effects on their periods, and that have the ability to entrain to daily light-dark and/or temperature cycle. In order to explore the circadian output pathway that regulates β-tubulin gene expression in Chlamydomonas reinhardtii, a nuclear transformation of the organism was performed with a β-tubulin promoter:reporter gene construct. To increase the efficiency of the selection, a cotransformation procedure was adapted with ble, an antibiotic resistance gene. To achieve maximum transformation efficiency, a cell wall-less mutant of Chlamydomonas reinhardtii was agitated in the presence of exogenous DNA, cell wall-digesting autolysin, glass beads and polyethylene glycol. Autolysin had been harvested successfully during mating of two different mating type strains, the optimal antibiotic concentration in the selection medium had been determined and other components of the transformation process were optimized. In successful transformations, a rate of 300 colonies per microgram of exogenous DNA or 1 transformant per 2 x 105 cells was obtained. Cotransformation efficiency varied from 1.3 to 3.3 % in successful cases. The transformants carrying the β-tubulin promoter::reporter gene construct will be used to investigate the expression of the β-tubulin gene by northern blot analysis. It will reveal whether this gene is regulated by the biological clock at the transcriptional level.

Name thesis:degree_name

Master of Science

Discipline thesis:degree_discipline

Department of Biology

Year

2002

Author dc:creator

• Khan, Mohammed

• Medical Sciences

Repository record dc:identifier

https://digitalcommons.wku.edu/theses/604

OAI identifier oai:identifier

oai:digitalcommons.wku.edu:theses-1607