Week 2- Getting the hang of it

Wow, I can't believe another week has already gone by! On Monday I checked up on the bacteria I was growing on plates and in liquid, with various mutations, to observe the role of certain genes in manganese oxidation. The plates provided me with a much clearer conclusion than the liquid, because hardly any of the tubes showed Mn oxidation in liquid. We also continued with the conjugation experiment. Unfortunately it did not go exactly how we had planned. We ended up with thousands of colonies of the pseudomonas putida containing the desired plasmid, but that is difficult to work with when we only wanted a few hundred so that we could select individual colonies. One of our three conjugation control plates did not look how we expected it to either. Its yellow color tells us that this E.coli plate had a P.putida contamination. My mentor, Kati, says we have to put the “re-” in “research” and do the conjugation experiment again before moving too much further. Kati taught me how to do the ancient art of “Replica-Plating” in order to move our P.putida from plates of one media on to plates of a different media so that the bacteria can be in the right environment to oxidize manganese. After all that, I made more media because we are going through the plates pretty fast!

On Tuesday I prepared for the conjugation experiment once again in hopes that I will end up with only a couple hundred colonies this time, rather than a couple thousand. Since I had done this process once before, and taken meticulous notes, Kati had me do the conjugation experiment much more independently this time. It really made me more confident in my abilities when I was able to carry out the experiment almost entirely off of my notes, with just a few questions here and there. Kati and I also re-ran the carbon source experiment where we are looking into the effects of various carbon sources on manganese oxidation in p.putida. This time we preformed the experiment in much greater detail by testing multiple mutants of p.putida, and remaking the stock solutions of the carbon sources. We yielded very fascinating results. There was no sign of Mn oxidation in the tubes containing glucose, but there was Mn oxidation in the tubes with xylan. This result fits nicely with my mentor’s hypothesis that p.putida might oxidize manganese as a way to help break down complex carbon sources, like xylan, to a point where it can consume it as food. We took picture of our results and my mentor might include them on a grant proposal she is submitting soon. It was really awesome to be part of an experiment that yielded the desired results!

I did a lot of “scrapbooking” on Wednesday, as my mentor and I like to say. On Tuesday afternoon and on Wednesday morning, I took pictures of the carbon source experiment and its exciting results! After taking all of the pictures in the photo room with the nice background and camera stand, I cut and taped in all of the photos into my lab notebook. I’m really glad we looked at the experiment when we did yesterday (only 2 hours into it), because the results were much more significant before many of the other tubes oxidized as well. I like having pictures to go along with my observations in my notebook, because Mn oxidation is shown by a color change, which is much easier to see than explain. My mentor took me in the next lab over to watch one of the lab’s primary investigators extract a shipworm out of a piece of wood from Alaska! It was so cool, and looked like fun! I attended a lunch seminar on goal setting. I liked it because sometimes I forget to take a step back and look at the big picture. Taking the time to think about my goals and how I plan to reach them really helps me with that. After lunch, I got back to my conjugation experiment. I suspended the bacterium that was conjugating on a plate for the last few nights into a media broth, and then I plated the different strains on plates that I am leaving out at room temperature for the night. This is the second time carrying out this experiment, and in hopes to not end up with way too many colonies again, I plated multiple concentrations of the conjugation. Hopefully one of the plates will work well, so I can make more of them!

Vanessa Green, the woman who makes this undergraduate intern program happen, took the interns to Astoria today! It was SO MUCH FUN! CMOP has a field station in Astoria that shares space with the Clatsop Community College. It was neat to meet the people there because they represented a different side of CMOP than I had been exposed to on the Hillsboro campus. These CMOPers were in charge of the hands-on, mechanical jobs such as collecting data and keeping their instruments calibrated and reliable. They collect data with a glider (a little underwater programmed airplane) that they send on missions in the Columbia River Estuary, and they themselves also dive down to the river floor to collect samples. I enjoyed being introduced to this important branch of CMOP. We had lunch at the Astoria column, and climbed all 164 stairs to the top for an awesome view! After that, we had a guided tour at the Maritime Museum. I had been there multiple times before but I learned more on this tour than any times before because our guide was like a walking history textbook. It was neat to learn more about the background and development of the Columbia River Estuary, since it is home to many of CMOP’s projects. It was really fun to go on this fieldtrip and get a more well-rounded sense of what CMOP does.

On Friday I continued my new conjugation experiment by using the replica-plate to copy my bacteria onto plates containing different media. I also checked on my first conjugation experiment and it seemed to work, although it was hard to tell with the thousands of colonies that looked more like one big smear. I saw a few oxidizing colonies on these plate (little brown specs), which told me that the mcoa gene (one of the two necessary oxidizing genes) was being expressed. This plate was made with the original strain that lacked both of the oxidizing genes. However, since some oxidation was being expressed, I know that the transposon disrupted the gene sequence encoding the negative regulator on the mcoa. YES! That’s just what I wanted to happen. I also continued with the carbon source experiment. Unfortunately, my mentor and I had a hard time quantifying the amount of protein in each of the tubes of various carbon sources, which makes quantify manganese oxidation unreliable. This is a glitch in the experiment my mentor and I will need to sort out soon.

That was a fun second week! I really feel like I’m getting more comfortable, and more confident!