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Week 8: Go the Distance

       Although it was an extremely long week, I too, just as Hurcules did, can go the distance. The end is nearing quickly, so I have to fit all of my experiments in.

      Starting on Monday, I got the results for the water and lignin plates I did last Thursday, and there was consistently a lot more growth from the samples in lignin, which is very promising. However, it looks like a lot of contamination occurred, so further tests were done to examine what happened. We didn’t know if it was a contaminant or a mutant. To test that, we streaked a known colony and an unknown colony onto lept, LB, and LB with (an antibiotic) ampicillin plates, because putida is resistant to ampicillin. So if it was a mutant, it would grow and if separate contaminant, it wouldn’t grow. Other results I was able to look at were from the MSTM and lept component experiment. What we saw was that having the yeast extract really helped in oxidizing manganese. But all samples were oxidizing, nonetheless, so I suspected that maybe there is a component in the minimal media that blocks the oxidation process in KG152. The final experiment I did on Monday was the plating of the sub-cultured experiment testing the different concentrations of lignin.
      Tuesday brought on some new experiences for me. This mostly comprised of the new protocol we used, called Semi-quantitative Reverse Transcription – Polymerase Chain Reaction (semi-Q RT – PCR). We used this very long protocol on our KG4 and KG152 strains within MSTM and lept medias to determine which proteins are being utilized more. Today’s part of the protocol had twenty steps, and lasted for most of the day. Because we are trying to isolate the RNA, we have to be very cautious with contaminants because RNA is extremely fragile. For the rest of the day, I replicated the experiment we did regarding all four strains in MSTM + water/lignin, because of the contamination that occurred.
      Wednesday was a very busy day. First, I looked at the plates that contained growth from the different lignin concentrations. And what we saw was very abnormal. There was more growth from the samples without manganese. But all plates had a steady concentration of pinprick colonies, which points to contamination, like we had in our last plate experiment. So we think that the contamination affected the normal growth pattern of our bacteria. Speaking of contamination, the results from the streaked unknown colonies could be analyzed. And, sure enough, there was no growth on the ampicillin plate, meaning it was contamination and not a mutant of putida. Next on this discourse, we wanted to find the source of the contamination, because even after extreme caution in the hood, the last two plating experiments have had contamination on them. We figured it might be the medias, so we plated MSTM, LB, and lept on lept plates because they’re the most visible. We then continued our RNA purification process, which lasted a few hours, and then stored for the next day. The rest of the day was spent creating a new experiment concerning the components of minimal media. The components of minimal media that differ from lept are ammonium sulfate, potassium dihydrogen phosphate, and sodium phosphate dibasic. So I used the three oxidizing strands with lept and these components to see if any interfere with the oxidation process.
      Thursday was mostly spent working on the RNA purification process. This portion focused on creating copy DNA from the RNA, and it lasted for about six hours. Then I looked at my samples in water and lignin, and most all of the oxidizing mutants were oxidizing, even in just the water. So because there was a lot of action even without a carbon source, I sub-cultured the samples, reducing some of the carry-over.
      Friday was a short day for me because my mom and I had to travel down to Corvallis to get some things straightened out with my living situation for next year, before they closed for the weekend. So I first looked at what results I had for the day. The results regarding the contamination source came out very strong toward the MSTM having a lot of contamination. Because that media doesn’t have a carbon source to fuel growth of the bacteria, it never grew large enough to become visible, therefore, going undetected until it was in an environment where it could thrive. Problem solved! But just in case, we also plated our newer batch of minimal media to make sure it was contaminant-free. Then I looked at my lept + MSTM component experiment, and two key observations were made. The KG51 strain had almost no oxidation from the lept + all three components, and KG152’s oxidation process was most affected by components containing phosphates, so these do act as limiting factors. The last experiment I worked on was the RNA purification process, which lasted a couple hours. The final thing I did was attend the final presentations for some of the interns who had completed their time here. It was good to see what I was expected to do for my presentation, and they all did very well! Then it was the end of the week for me. And with that, it was an end of an era. An era of my first research notebook: filled. It was time to get a new notebook for the remainder of my time here.