It has been a difficult semester so far. Preparing for graduations and renovations in the labs have made performing experiments extremely difficult. At this point of my research project I have put a hold on my transposon mutagenesis project and have focused more on my Opp project. With this project I am trying to see if glutathione affects a BCG mutant that has a ABC transporter knocked out. I will be expressing this mutant to 0,4 and 8mM glutathione while measuring its optical density and viability over 4 days.
This experiment is somewhat difficult because Opp needs to be exposed to streptomycin and kanamycin antibiotics to ensure that the mutant plasmid stays within the bacteria. Giving it these antibiotics however retards the growth of the organism and makes it rather difficult to have mid-log growing bacteria at hand . After this project is completed and I solve my refrigerator problem then I will pick up back on the transposon mutagenesis work .
I also plan on using a thiol fluorescence’s kit to measure the amount of glutathione present in the cytoplasm’s of BCG at 0, 4 and 8mM. This test will also be conducted on persistent BCG using the Wayne model. The Wayne model comprises of using nunc tubes to limit the oxygen available for the bacteria, while growing it for 100 days until it is in a environment of low nutrients and high hypoxia.
Hopefully I will be able to complete these three experiments before I graduate and obtain statistically significant data which will help us to better understand the effects of glutathione.