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Week 4- The Swimming Test for P.putida

It turns out that streaking for single colonies of bacteria is even trickier than I thought! Monday is the third time I’ve streaked the mutants obtained from my conjugation experiments I did over the past few weeks. I now have 30 potential mutants where Mn oxidation has been restored, out of around a couple hundred thousand colonies of bacteria. In these oxidizing colonies, my mentor and I hypothesis that the transposon disrupted the gene encoding the negative regulator because of where it randomly inserted itself into the bacteria’s genome. Some of the mutants appear to be oxidizing more, and may turn out to be more promising as the project progresses. I am repeating the carbon source experiment with a few revisions this time around. On Monday I started the overnight cultures for the experiment, and included another strain of P.putida to look into because of its proposed potential to degrade lignin. The other experiment that I began this week relates to the negative regulator identification project, involving my mutants. My mentor thinks that there could be a link between flagella production and Mn oxidation, so we will test each of the mutants on motility plates with a few controls. To start preparing for this experiment, I took the three control strains out of the -80 degrees Celsius freezer and streaked them out onto a plate so they would be healthy, growing, and ready to go for the rest of the week.

 

Since CMOP is moving from OHSU west campus to Marquam Hill in the fall, everyone has begun preparing for the move. To help my mentor store some of her P.putida strains, I mixed some of the overnight cultures from Monday with glycerol and put them into vials that will be stored at -80 degrees Celsius. The glycerol is used as an anti-freeze so that ice crystals won’t grow and puncture the cells. I am weaning my bacteria off of rich media each day this week until they are near the point of starvation, because we are interested in the possibility of bacteria oxidizing Mn as a way to help it digest food in this stressed condition. Of my 30 potential mutants from Monday, only 4 had formed single oxidizing colonies. I created overnight cultures with those colonies for upcoming experiments. I also prepared some stock solutions and media that we were running out of, and they were ones I hadn’t made before. One of the media is for the motility experiment, and involves much less agar so that the bacteria can “swim” through the media on the plates.

 

My mentor and I are taking a different approach to the carbon source experiment this week. We are culturing the wild type of the bacteria in a flask rather than a test tube, and periodically measuring the optical density of a flask with no food and a flask with lignin as the only food source for the bacteria. We also added Mn(II) to both of these flasks since we are interested in the role of Mn oxidation for breaking down and digesting complex carbon sources. We predict that there will be no cell growth in the flask with no food (since the bacteria is starving), but we hope that there will be growth in the flask with lignin because this would tell us that the bacteria is able to consume lignin with the help of manganese oxidation. The optical density test, executed with a spectrophotometer, will tell us “how cloudy” the solution is, and therefore how much cell growth has occurred in the flask. Returning to the other experiment with my mutants, I plated them on the motility plates I made on Tuesday. I am observing and measuring how much each of the mutants “swim” on these plates compared to my controls of known motile and non-motile strains. The purpose of this experiment is to learn more about my mutants by quantifying their motility, and therefore their flagella.

 

On Thursday my mentor’s grant proposal was due to the National Science Foundation. She has been working really hard on this proposal for as long as I’ve been an intern, and I’m sure many weeks before that. She is proposing a project revolving around the degradation of lignin by means of Mn oxidation in Pseudomonas putida. The carbon source experiments that I have been doing serve as some of her preliminary data. In fact, she included the photos I took of my data in her proposal! It’s pretty cool to be involved in a project that has such potential. I offered to read over her proposal for a final spelling and grammar check. It was really impressive to see all of the information she included in the 15 pages of background, data, and broader impacts of the project. I learned more about the work I’m doing by reading it because it put everything into a broader context. On Thursday I also continued with my motility experiment by making more media, pouring plates, and culturing the rest of the mutants. For the first motility experiment I only used 4 of my 30 mutants because those were the only ones that had begun oxidizing at that point. Since the mutants had been growing on the plates for a few days by Thursday, all of the mutants were oxidizing. It turns out this motility experiment takes a lot of careful timing in order to see anything significant. By the time I got to the lab on Thursday, a lot of the mutants had already “swam” through the whole plate and reached the edges of the plate. This doesn’t tell me much involving the motility phenotypes of the mutants, which is why I would’ve liked to check on them earlier (but that would’ve been in the middle of the night…). I prepared the cultures and poured the plates on Thursday so they would be ready to go Friday morning and hopefully be doing something interesting on the motility plates by Friday afternoon. I also transferred my mutants off of Lept media onto LB media plates, which is much richer, so that my mutants would stay healthy and happy. I did another spectrophotometer test on the two flasks I mentioned yesterday, and I got really exciting results! There was a significantly higher absorbance in the flask with lignin, which means there was more bacterial growth in this flask than in the other. Therefore, the bacteria must have been able to consume lignin for its food source.

 

The first thing I did when I got to the lab Friday morning was plate the rest of my mutants (from their overnight cultures) onto the motility plates I made on Thursday. This motility experiment is really time sensitive, so I’m still playing around with when is the best time to start the motility assay so that I will be here when the bacteria are doing something cool. By the time I left Friday afternoon, quite a few of them had begun swimming. I also looked at my motility plates from the first time around, and a lot of them had oxidized. I sorted my mutants into categories of motile and non-motile, oxidizing and non-oxidizing, so that I can group my mutants by their phenotypes. I ran another optical density test on my cultures that I have in the flasks with and without lignin, but Friday’s results were trickier to interpret than Thursday’s. The absorbance levels in both flasks decreased. I am guessing that the flask with little to no food has now run out and is dying. As for the flask with lignin, I see a thick biofilm on the glass which throws off the cell density measurement since a lot of the cells aren’t in solution. The media the strain was inoculated in contained glucose, and although I did starve them for a while, there might have been a little glucose left in the culture before mixing it with the other flask components. Therefore, the bacteria may have just been eating the little trace of glucose that was in the flask, and are now left with the challenge of eating lignin.