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Week 9: Circle of Life

       As the final week approaches, we start to round out our time here at CMOP.

      The first thing we did on Monday was run the PCR we worked all last week to prepare. The results brought about some mixed emotions. The good news was that there was no detectable DNA contamination, and KG4 is expressing MnxG, which is what we were looking for. But the bad news was that KG152 was not expressing MnxG like it should. It is worth considering, however, that the cells were getting slightly old and dying, and therefore, might not be producing much of that protein at that time. There were some very promising results, however. After examining the second batch of MSTM for contamination, it was proved safe and sterile. Phew! The days of all of the interns were then put on hold to attend a focus group regarding our experiences over this summer. Although it lasted a lot longer than I expected, a lot of good thoughts and observations were discussed, so it went by pretty fast. Going back to work, I also looked at how the experiment testing reactions to lept + MSTM components, and results were pretty similar to Friday. My main conclusion was that the absence of yeast extract and the addition of the phosphates contribute to very little to no growth in that minimal media. I then looked at my water and lignin sub-cultures, which were looking more realistic. So I plated them, in order to assess their growth.
      There weren’t any results or experiments to run on Tuesday, so I spent the entire day hand-sorting and analyzing over 5000 data points from the lignin fluorescence experiment I ran a few weeks ago. Although I already reported the outcomes in my earlier blog, the data was all compiled over the course of this week.
      On Wednesday, I had some results from my water and lignin plates. After two days, there was more growth in the oxidizing mutants than the non-oxidizing mutants, suggesting that although the non-oxidizing mutants can live off of lignin, the bacteria would prefer the use of manganese oxidation. There was also a lot more growth in the lignin compared to the water, meaning putida can break down lignin and use it as a food source. I then used the rest of the day to analyze the fluorescence data. And by the end of the day, I had a graph! All of my data was looking good, but because of a consistent, outlier point, some of the data was skewed. So my project for Thursday was to discard that point to see how it affected the remaining data.
      And that is exactly what I did. I got rid of the outlier point, and my data came to support our hypothesis! It was the first time we could actually see and quantify lignin being broken down, so it was very exciting. After that, we set up two more PCRs, using our cDNA from our previous experiment to answer a couple more questions. We wanted to know if each sample had roughly the same amount of cDNA present. So we amplified the “housekeeping” gene, which is a ribosomal protein, whose levels should be the same under most growth conditions. And then we also wanted to know how the expression of McoA responds to MSTM + glucose and lept medias, so we amplified that gene. Lastly, I got some bacteria out of the freezer to set up for a repeat of the lignin fluorescence experiment, to confirm the results obtained from my data.
      On Friday, we finished up the two PCRs, and we came to the conclusion that there are relatively the same numbers of cells, there weren’t any McoA genes being expressed (which is good because they were deleted), and there was more McoA in the lept media. Then I continued to set up bacteria for the lignin fluorescence experiment to be performed on Monday. Lastly, I discussed my project with Kati and the direction I should go in my paper and presentation.