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Week 6: Hakuna Matata

       This week was very different from weeks in the past. That is because Kati, my mentor, was on vacation the whole week. It was a very relaxed week, because I had the freedom to choose which experiments I wanted to run and I could do everything on my own time. Yet it was also one of my busiest weeks because I wanted to run a lot of different tests. I was also not here on Friday, due to a family wedding, and so I needed to fit everything into four days.

      I started my week with a project with Rick Davis, another mentor in my lab. Together we attempted to track lignin degradation by fluorescence, using a protocol that he was familiar with. We used the wildtype and KG180 strains of putida, E. coli as our negative control, jostii as our positive control, and no cells as our baseline control. The media the cells were in was MSTM, and we also used two sets of everything – one with manganese and one without. After an hour, there were positive trends, which was promising. So we left it running for 24 hours, reading every ten minutes. Then I created an experiment testing how the cells grow with different concentrations of lignin. The setup was the same as usual using the wildtype strain, but I used 0, 2.5, 12.5, 25, and 50 microliters of lignin.
      After 24 hours of running the lignin fluorescence experiment, our results took a turn downward. We had downward trends that were all very similar in rate. So we made the decision to try again on Wednesday. But there was a lot to be done on Tuesday. I started an experiment testing how the presence of oxygen (or lack of) affects which oxidizing proteins are activated within our cells. So I used all four strains with Lept, MSTM with lignin and water plates as media, because that’s what we had left. I was able to restrict the amount of oxygen present by using a GasPak for multiple days. I then started another experiment testing how my cells respond to light and dark on solid media. Again, I used all four strains and lept, MSTM + glucose/lignin plates for media.
      On Wednesday, Rick and I got together again and ran the lignin fluorescence experiment. This time, however, we added glucose into the media as well in order to kind of “wake up” the cells to get them activated and eating. After an hour, there were downward trends, but we chose to keep the test going to see what happened over time. That took up a lot of the day, so for the last half, I was just able to re-culture an experiment from last week that utilized the different carbon sources.
      Thursday was mostly for results. The experiment with different concentrations of lignin was all oxidizing, suggesting carry-over. The experiment regarding different oxygen levels had the KG4 and KG51 oxidizing in all medias/oxygen conditions, except for the MSTM + water, and neither KG152 nor KG180 were oxidizing, except KG152 in lept. After two days of the light vs. dark experiment, the results were pretty much the same as the oxygen experiment. But the most exciting results were from the lignin fluorescence experiment. Although all of the data had downward trends, they were all going down at different rates. In fact, the bacteria that is supposed to be degrading lignin is not decreasing as much as the ones that aren’t supposed to, so that is very promising. And after hours and hours of compiling and analyzing the data, it worked! I produced a graph that proves that the wildtype strain degrades lignin as well as the jostii, a known lignin degrader, in the presence of manganese.
      With that good news, I enjoyed my weekend at the beach celebrating an amazing couple!