Weeks 8 & 9

These weeks were spent doing a variety of evolution and growth curve experiments with the bacteria on a minimal media (M9) with ethanol. My first growth curve was successful, as I was able to show that the absorbance readings, a measure of the number of bacterial colonies, went up steadily each half hour throughout the 12 hour period during which I took readings. After seeing the growth curve work with small amounts of LBS present, I took on the next step, which was introducing the bacteria to M9 media without the presence of LBS. I used a new technique for this which involved washing the cells that removed any LBS media left in the culture, and tried to grow the bacteria on the M9 this way, but this led to the bacteria being unable to grow on M9 at all. After meeting with my professor about this, he discussed several reasons why this might occur, namely that the centrifugation required for the washing process was too severe for the bacteria, particularly since their membranes were being agitated and they were then being placed into a media containing ethanol, which contributes further to the destruction of their membrane. Another option might be that the bacteria need to be evolved to withstand a minimal media with ethanol in it first, so the extra LBS in the media might have been facilitating this evolution. To figure out which of the two options was more likely, I inoculated strains in M9 minimal media straight off the plate and tried to perform growth curve experiments with the strains using the minimal media starter cultures as well as the LBS cultures that were washed. Though nothing grew in either condition, my PI advised me to try evolving them over longer periods of time (re-culturing either every 12 hours or every day) to see whether the bacteria would grow when re-cultured. If they do, this will provide a more straightforward approach to use to evolve the bacteria to ethanol. If they do not, I will need to continue introducing them into the M9 starting with LBS, evolving them to survive and be culturable in M9 media.

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