Measurements of DNA Damage and Repair in Bacillus anthracis Sterne Spores by UV Radiation.

摘要

Spores of Bacillus anthracis (Ba) sterne were irradiated with 267nm UV light using small light emitting diodes. The pRB373 plasmid with a red fluorescent protein was transformed into Ba sterne cells prior to irradiation. Following irradiation, germination media was added and the spores were incubated for various times, to allow for DNA repair. The pRB373 plasmid was isolated and analyzed using real-time PCR. Primers were designed across the RFP in the plasmid yielding two amplicons, 245bp and 547bp long. PCR amplification was not achieved for germinated samples. Spore samples isolated using bead beating methods were amplified. Results indicate a quicker amplification (lower Ct) for irradiated samples then for un-irradiated. Lack of PCR amplification in germinated samples is attributed to too damaging an extraction method for Ba cells. This observation was not expected. Ba Survival Curves were also developed using the quadratic fit y = alpha x + beta x squared. Averaging results form 3 experiments, alpha is reported as -0.0144 + or - 0.008 and beta as -0.00001 + or - 0.0002. Actinometry experiments corrected for the efficiency of the LEDs in all experimentation. Fluorescence measurements monitored germination and outgrowth; they indicated a delay in germination of irradiated spores. AFM images showed morphological changes in irradiated spores.