Week 8- At last, the cells arrived!

On Monday I did not get as much information from my motility plates as I would’ve liked, but I did get a few good photos. There was denser and darker oxidation occurring next to certain carbon sources. More oxidation means that there are more cells, and if there are more cells, there must have been more growth in that area. Another way to observe the bacteria seeking and eating the carbon source was when there was a bulge in the circle towards a certain carbon source. This means that the bacteria made flagella so they could swim more towards the food source they could eat. One thing that might not have been ideal was that the inoculated cells I used were close to a day old, so they might’ve already reached late stationary phase. Also, the motility plates could have dried out this weekend before the bacteria swam to its full potential. If I want to do this experiment again, I will make these revisions.

The “quick and dirty” method of making competent cells last Friday and trying to do control transformations failed yet again; there was no growth on any of the plates. However, this failure does not tell me much since the cells were prepared the “quick and dirty” way.

I learned how to do something new on Monday! Since the competent cells we ordered last week hadn’t arrived by Monday, it was another day of finding something to do while we wait. At the beginning of my project, I identified 28 colonies where the original mutation on mnxR (deletion of the positive regulator of the two oxidizing genes) was suppressed and oxidation was restored. To prove that these colonies weren’t just random contaminants of wild type, I ran my first ever Polymerase Chain Reaction (PCR). I prepared my samples of genomic DNA with the PCR machine, and then ran them on an agarose gel next to controls that either contain or lack the mnxR deletion. I found out that two of my so-called mutants didn’t contain the mnxR deletion…which was the strain that I used to do my conjugations with in the first place…

I struck out all my 28 mutants on LBampkan (contains ampicillin and kanamycin) to see if they are just wild type or if they are one of the delmnxR mutants. If the mutant grows, that means it’s delmnxR because it would have taken up the plasmid (that contains a kanamycin resistant gene) during the conjugation. However, if the mutant does not grow, then the “mutant” is actually just a random wild type contamination that did not get conjugated and therefore does not contain the plasmid.

On Tuesday I attended a lab meeting where a few of the undergraduate interns who are close to being finished presented their summer research; I will do the same in a few weeks. It was fun to see what other interns are up to and to see how all of our projects are closely related because of being in the same lab.

My mentor and I were really bummed that the cells didn’t arrive on Tuesday and also were getting a little nervous since this project is running a tight timeline. Since I didn’t have my cells to work with, I did some housekeeping such as make plates for my mentor and feed my carbon source experiment. I also checked on all my mutants I plated on LBampkan on Monday. 25 out of 28 mutants grew, which means that the majority of them took up the transposon during the conjugation and were consequently kanamycin resistant. The other three were either wild type contaminations or revertant colonies where they kicked out the transposon because it was making them sick. On Tuesday I also had some time to work on my final paper that is due at the end of this internship, so it’s nice to get started on that now!

As I was waiting for the cells to arrive on Wednesday, I set up a few new experiments to differentiate the 28 mutants by their phenotypes for growth at various temperatures and biofilm formation. It was a tedious set up since it involves 40 tubes… but thank goodness for master mixes! I have to wait until Monday, when they’ve reached stationary phase, to analyze the tubes with a crystal violet stain and photos. I also made more media on Wednesday since I went through so much of it with these two phenotype experiments.

***On Wednesday afternoon my competent E.coli cells arrived at last. Yippee! I transformed the ligated DNA of 10 of my mutants. I followed the protocol that came with the cells, which involved heat shocking for very precise amounts of time. I hope my transformation works this time!

I fed my carbon source experiment on Thursday and took some photos. The biofilms are consistently thicker in the tubes where Mn(II) oxidation is occurring in the presence of a complex carbon source. To make sure the clumpy brown biofilm I saw was indeed Mn(IV) oxides and not just clumps of lignin or humic acids, I dropped LBB onto residual biofilm matter after transferring tubes. The reaction turned blue, which LBB does in the presence of Mn(IV) oxides. Additionally, the tubes with the thick biofilms had clearer solutions, which perhaps imply that the complex carbon source has been broken down. I’m hoping that this experiment will get to a point where my photos will be strong enough to support our hypothesis.

Late in the afternoon on Thursday I inoculated all of the colonies that popped up on Wednesday’s transformation plates in LB with kanamycin (so nothing unwanted would grow in the culture). Unfortunately there were only 7 colonies total from 16 plates. I was hoping for more colonies, but now I guess I’m just hoping that these colonies really contain the plasmid.

With only 100microliters left of competent cells, I decided to do another transformation with two of my mutants since Wednesday’s transformations were not as fruitful as I wanted. Before I could do this transformation though, I needed to make more LBkanamycin plates. With so many things going on at once made for a busy couple of hours, and a lot of timers set!

First thing Friday morning I isolated plasmid DNA from the 7 cultures I inoculated Thursday afternoon. I used the QIAprep Miniprep kit for this isolation, which included a page of protocol and all of the necessary buffers. I had used this kit once before, but this time it felt much more natural. After I isolated the plasmid DNA, I took it over to the Nanodrop which quantifies the DNA. The concentrations were plenty high and the protein to DNA ratio was right where it should be for all of my samples; yahoo! Then I prepared my samples for sequencing by filling out a sheet of information about my samples and including the primer I want to be used. It was really exciting to send of my samples to be sequenced; it feels like I am one step closer to where I want to be in my project!