Week 7- The Waiting Game

The culture tubes I started last week with various P.putida mutants growing in the presence of various carbon sources did not much change from when I checked them on Friday. This makes me wonder if maybe there is a limit on how much of the carbon source the bacteria are capable of breaking down and digesting.

On Monday I learned how to do something new: run a “gel electrolysis”***. I am using this technique as a way to check that the plasmid DNA I isolated last week really is plasmid DNA, and not just genomic DNA. This technique involved preparing an agarose gel and injecting my extracted plasmid DNA (mixed with dye and digested with an enzyme called PstI) into the wells of the gel. Then I ran an electric current through the gel for about a half an hour until I could clearly see separated bands on the gel, which represent different fragments of DNA. Unfortunately, my results were a disappointment. My so-called plasmid DNA turned out to contain no DNA at all. My error either lies in the ligation or the transformation step. I am running three control transformations to make sure that the competent E.coli cells I made really are competent, which would eliminate this step from being a suspect for my non-existent plasmid DNA. These cells are somewhat tricky to make, but too expensive to order, so it’s important to verify their quality. As a way to test their competence, I transformed them with two known plasmids and also in the absence of a plasmid. Both of the plasmids used are kanamycin resistant, so if my cells really are competent, they should take up the plasmid and grow on the kanamycin-containing plates.

I completed the ethanol wash on the digested DNA and carried out the ligation. I’m worried that the ligation might have been the faulty step in the process the first time around, so I used someone else’s stash of the enzyme and buffers because the one I was using before might have expired.

The second round of transformation plates were the ones covered in itsy-bitsy colonies. To see if all of those colonies were actually kanamycin resistant and not just random mutations, my mentor Kati streaked out a swab of colonies those plates onto LBkanamycin plates on Friday. There was growth on all of the plates, but some of the colonies looked healthier than others. I inoculated a colony from each plate into LB (rich media) and kanamycin to see if the colony really is kanamycin resistant (i.e. if it took up the transposon).

To start with the bad news, there was growth on all three of my control transformation plates, even the one without a plasmid… This tells me that a source of error lies in the competent cells, or in the transformation I did with these “competent” cells. One of the media I use in the transformation process, LB, looked contaminated on Monday so I threw it out. My mentor and I realized that I had used that batch of LB for the transformation process. So, on Tuesday I made a new batch of LB liquid, among other media and plate needs, and used the new LB as I repeated this control transformation experiment. I hope that it was just a media contamination and that my competent cells are ok so we don’t have to buy them.

            For a little more bad news, only one of the 6 colonies I inoculated in liquid media and kanamycin Monday afternoon grew. This means that none of the colonies that were growing on the transformation plates had really taken up the plasmid, and were actually just random mutations. For the one tube that had growth, I centrifuged a few milliliters of it and saved the pellet in the -20 degrees freezer so that I can isolate the plasmid DNA in the future. I don’t want to do this task just yet because it’s a tedious process to do for only one sample.

            On Tuesday I began “feeding” the carbon source experiment cultures that seemed to have hit stationary phase this weekend. Since we think there is a limit on how much the bacteria can consume at a time before the carbon source becomes poison rather than food, I will periodically “feed” them. On Tuesday I separated the bacteria from the old media with a centrifuge and then fed the pellet with new media, Manganese, water, and a carbon source of interest. As bacteria hit stationary phase, some are dying and changing the nature of the media, which is why I want to replace the food source periodically rather than just adding more to the existing media.

Yet again, my control transformations disappointed me. There was growth on all of the plates when there shouldn’t have been any on one of them, which tells me that my troubles were not a result of contaminated media, but rather incompetent cells. Ugh. As an alternative to using the “competent” cells I tried to make, my mentor dug out two other strains of E.coli that have the potential of becoming competent cells. However, neither of these strains are in a ready-to-use condition. One of them already contains a plasmid and the other one lacks thymidine, which is needed to replicate DNA. On Tuesday I struck out both of these strains onto plates to get them growing, and did a control by having one plate lacking thymidine. Surprisingly both strains grew in both conditions, so something is wrong with these saved strains since the strain lacking thymidine should not have grown on the plate lacking that essential component. Since the summer is only so long and all of these “competent” cells have been giving us such trouble, we decided to place an order today and just buy them. On Wednesday I made four types of media to restock our shelves. One of the media I made was for MSTM motility plates, which is a minimal media containing only a tiny amount of agar. I’ve done a motility experiment once before this summer, and its purpose is to see how the bacteria swim. Last time I used motility phenotypes to differentiate between my mutants, but this time I’m just looking at the wild type, providing it with Manganese, and dropping various carbon sources on the plate as well. I want to see if the bacteria swim towards carbon sources such as lignin in its minimal media condition. If they do swim towards lignin that would mean that they are using their energy to create flagella to swim towards the food source. I played around with how far the carbon sources are dropped from the inoculated cell drop because I wouldn’t want my bacteria to die before they even reach the carbon source.

On Thursday morning I checked on the motility plates I made Wednesday and didn’t see anything exciting. The inoculated cells I used on the plates had been growing for 20 hours, my mentor pointed out, so they might have already reached stationary phase by the time I plated them. I sub-cultured my cells early Thursday morning so that they’d (hopefully) be in their exponential growth phase by the afternoon. I made another batch of motility plates because they have to be used the same day they’re made. One more revision I made to this experiment was that I dropped the carbon source closer to the cells. I did this because with the presence of Mn oxidation but lack of swimming I observed on yesterday’s plates, I think the cells were starving before they even got near the potential food source.

Since the control transformations turned out so strange on Wednesday, we decided to do an experiment on the cells to see if they really are what their label says. The reason we have some doubt about this is because they are not cells we ordered or made; instead, they were sent to us from another lab. There is a chance that the various strains got mixed up when the other lab was sending us these cells. The E.coli strain lacking thymidine grew in the absence of it on Wednesday, which either means these cells aren’t what we think they are, or that we were testing them in too rich of a media so they didn’t necessarily need thymidine. To narrow down this question, on Thursday I inoculated the strain into medium of varying richness with and without added thymidine. I also did the same with the other strain of E.coli that already has thymidine for a positive control.

The carbon source “feeding” experiment I have going on seems to be working well so far since there appears to be more growth and Mn oxidation since I fed them. The biofilms look much thicker in the oxidizing cultures than they do in the non-oxidizing ones, which supports the hypothesis that P.putida uses Mn oxidation to help it break down and digest the complex carbon source so it can eat and grow. However, along with all of the other experiments I have done for this project, it’s very tricky to quantify this observation.

Friday morning I went on a tour of the primate center. It was so cool hearing more about all of the policies that this facility deals with, and how it was established. I feel like I have a much better understanding on why animal testing is necessary for medicine to advance. It was also pretty cool to see so many teeny tiny baby monkeys! At lunch on Friday, there was a brown bag seminar on graduate school. I enjoyed hearing the women talk about how they chose their program, what a typical day is like, and how much they love it. My lab also had an impromptu 2 hour lab meeting since our primary investigator just got back into town this week, so we got to hear how everyone’s research is going in a roundtable. By the time all the tours and meetings were over, I still had a lot of work to do!

I made observations on my motility plates and was excited to see that there was a distinct difference is swimming and Mn(II) oxidation densities within the motility plates. As of Friday, the only increased swimming was towards glucose, which is pretty obvious since it’s a simple carbon source that’s easy for them to eat. On Monday I’m hoping to see something more exciting, like swimming towards lignin!

I did the “quick and dirty” method of making of competent cells on Friday and then transformed them. I did this because my mentor and I want to know what’s going wrong and why these cells aren’t taking up the plasmids. Even though we will have commercially made competent cells next week (hopefully), we want to know why the strains we have aren’t working, so we are repeating the process over and over again.

I also “fed” my carbon source experiment on Friday, so hopefully P.putida will be growing happily and healthily in the tubes with the complex carbon sources over the weekend!