Week 6- Putting the "re-" into "research"

The Carbon Source Experiment I started last week (many identical cultures growing and plating dilutions every couple of days) involves a lot of repetition repetition repetition. I start the day with counting colonies on the dilution plates from the other day and calculate colony forming units (CFU) per mL. Then I make all of the same dilutions as I did before, but with a different set of cultures (that have had more time to grow), and I plate two dilutions for each. After I counted colonies Monday morning, I had two sets of CFU/mL data, so I could compare growth in the presence and absence of lignin. Although my data did imply that more growth had occurred in cultures with lignin as the only food source than in the cultures with no food source, all of the CFU/mL values were within the same magnitude so there really wasn’t anything too significant, yet. I go through 8 plates each day that I plate dilutions, so on Monday I made media and poured plates; this will probably become a daily routine. In regards to the other project, the transformations I plated on the LBkanamycin plates last week showed no signs of growth, which is pretty disappointing. I had hoped that some colonies would have taken up the transposon (that contains a kanamycin resistant gene) and grow, because I want to select those transposon-containing colonies for my upcoming experiments. I’m hoping that this process is just being slow, and that there wasn’t anything wrong with the rest of the experiment. I put them in the 37 degrees Celsius incubator in hopes that the temperature would help speed up growth. Last week I grew up all of my mutants on plates and on liquid to observe any discrepancies between the two for each mutant. I found that some mutants oxidized better in one substrate than the other. On Monday I took photos of all of the plates and culture tubes because pictures show the differences better than words.

Tuesday morning I counted colonies and saw that there were no significant differences in CFU/mL values, so my data doesn’t support nor reject the hypothesis that P.putida can consume lignin. Since my transformation plates were bare Monday, and by Tuesday only 3 out of 10 plates had even a single colony, my mentor and I think that maybe there wasn’t enough gDNA to begin with. To check if these colonies really did take up the transposon, I inoculated them in a broth containing kanamycin. If there is growth in the overnight culture tube, I know that the colony really did take up the transposon. I quantified the digested DNA with the NanoDrop and found that many of my samples contained very low concentrations of DNA, so we’ve decided to do this whole process over again. With the concentration calculated by the NanoDrop, and knowing that our experiment would be ideal if we started with 100ng of digested DNA, I can calculate the volume of the digested DNA I will need to start with from each mutant. In order to have all the materials we need to repeat the experiment, I need to make more “competent” E.coli cells (cells that are capable of taking up a plasmid in the transformation step). This process takes two days and involves a lot of centrifuging, waiting, and incubating on ice. Based on the concentration values from the NanoDrop, some of the mutants must be restarted from the restriction digest step because they contain hardly any gDNA, while other mutants have enough gDNA that they only need to go through ligation again. On Tuesday I completed the ligations I needed to repeat, which involved mixing the digested DNA with a ligase enzyme.

It feels pretty busy right now because I’m going back in my experiment and re-doing different parts for various mutants. To transform the ligated DNA I prepared on Tuesday, I had to create competent E.coli cells. This involved an overnight culture, and a lot of heat shocking. For the other mutants that had hardly any gDNA to start with first time around, I had to take them all the way back to the restriction digest step (which involves mixing the gDNA with a specific enzyme). With a kit that explains the entire protocol and includes all of the ingredients, I isolated plasmid DNA from the colonies that actually grew on the LBkanamycin transformation plates. Since my data from counting colonies on the carbon source dilutions plates haven’t been showing too much, I think the ascorbic acid might not really be dissolving the Mn oxides like I want. When these clumps are left in solution, they can really mess up my data because if I accidently plate a clump, there will appear to be tons of cells in that culture when that’s not really an accurate representation. In response to all this, on Wednesday I treated 3 sets of my cultures with either ascorbic acid, LBB (a blue colored acid), or Triton X-100 (a detergent) to see which did the best job of “dissolving” the clumps.

On Thursday morning I began with counting colonies on my dilution plates and decided that the ascorbic acid seems to be the best treatment for the oxides, but should be used in a higher concentration. As a different way to approach quantifying the amount of growth in my culture tubes (P.putida growing with no food vs. lignin as only food source), I did a crystal violet stain. The stain is a beautiful color, but also contains phenol so I had to be pretty careful with wearing gloves etc. This method of quantification is pretty subjective to the naked eye, but the cells were stuck to the glass all over and it would’ve been too tricky to re-dissolve them for an optical density test. Instead, I took photographs. I also made a lot of media and plates on Thursday because the dilutions I do every day use quite a few of them... I plated another set of dilutions, but did duplicate of each make my data more reliable. Going back to one of the first approaches I took with the carbon source experiment, I started cultures of various mutants of P.putida growing in the presence of Mn and various carbon sources such as cellulose, xylan, lignin etc. to see where the best growth occurs and how Mn oxidation contributes. In preparation of ligating the restriction digests, I washed the digests with ethanol to get rid of any residual enzymes from previous steps of the experiment.

Early Friday morning I counted colonies and saw more of the results my mentor and I were expecting, so the high dosage of ascorbic acid must have dissolved the clumps pretty well. The wild type of P.putida grew better (more colonies on the plate) when lignin was its only food source than when there was no food source. This result fits with the hypothesis that P.putida can use Mn oxidation to help it break down and digest complex carbon sources. On Friday Vanessa, the undergraduate internship coordinator, took us all on a fieldtrip to the Gorge! It was sunny and beautiful. We stopped at the Vista House, the fish hatchery, went on a tour of Bonneville Dam and then hiked to the top of Multnomah Falls! Even though I’d been to all of these places before, I learned a lot because of the tour guide. I also hadn’t hiked up the trail to the top of the falls before, so it was fun to do something new! The internship program through CMOP is so well-thought out, and I think it’s great that they factor in fun fieldtrips like today’s to give the students a bigger perspective on CMOP’s projects.