The Eighth Week – Busy Bee

My internship is winding down, and I’m finding that sometimes I really dread coming to work. Now, that might sound like a terrible introduction to a blog entry written on the website of my employer, but it’s true, and I know everyone has felt that way before. It’s important to identify, accept, and cope with a negative attitude. My dread has stemmed from repeating what seem to be mindless tasks and spending much of my in between time waiting for bacteria to grow, get cold, oxidize, take up DNA, or a multitude of other things. I didn’t feel like I was doing anything with a purpose or using my time effectively. This week, I resolved to change that and really enjoy what little time I really have left here! I have done that by staying busy; like a worker bee. As long as I’m busy doing something the work doesn’t drag on, and most of the time I really enjoy what I’m doing.
 

The experiments that have required the most time, and made me feel like I was wasting that time, are the ones to alter the DNA of P. putida through PCR, digestion, ligation, and transformation. The protocols for many of these processes don’t really make any sense, and are sometimes quite arbitrary with long wait periods. For example, making cells competent for a transformation, which I have done several times this week, requires everything to be kept cold while you suspend the bacteria in calcium chloride. But we don’t know why keeping cells cold and putting them in calcium chloride makes them more likely to take up DNA in the form of a plasmid when heat shocked for 30 seconds at 42°C. But that’s what you do. 
 

This week, I began by digesting my second PCR product, which is just adding the PCR product (a piece of DNA) some restriction enzymes and buffers and incubating for an hour and a half. Then I ran that digested PCR product on a gel and cut out the band of DNA I wanted just like I did last week with the pUCP22 and first PCR product. Using a special kit called the “Gene Clean turbo kit” I isolated the DNA. This is another one of those silly protocols where you just trust that it works. I performed a series of short spins with the DNA product and a salt solution through a filter that evidently the DNA magically sticks to very well. Then, you add the “elution solution” –which is really just water–let it sit for a while to let the DNA loosen up a bit, and spin it down again and magically the DNA is removed from the filter and left in the tube. Once this is finished we ran the final DNA through a gel to make sure we still had some left, and then we began the ligation and transformation. Quick Ligase is basically DNA glue that works really quickly. To perform the ligation I just added the pUCP22 plasmid (digested so it has a hole) and the PCR product (or piece of DNA we want to be on the plasmid) along with some buffer and the quick ligase and let it sit for 5 minutes. During these 5 minutes the ligase does its magic and glues them together. So now, supposedly, we have a mutagenized pKG207 gene (that was mutagenized during the polymerase chain reaction or PCR) stuck into a plasmid that we are going to try to put into E. coli before moving to P. putida. This is the transformation reaction, where I put the plasmid from the ligase reaction and some competent cells together and hope the cells will take up the plasmid when heat shocked. Another, cross your fingers and hope it works step. Once they have been heat shocked, I added some SOC (really good bacteria food) and incubated them so they could recover, before plating them on LB plus gentamicin plates. We plate them on gentamicin plates because the plasmid encodes for gentamicin resistance, so only the cells that took up the plasmid will grow.
 

The first time I did all of this with my first PCR product and I used competent E. coli cells, which just means I used some already competent E. coli cells instead of making them myself. And all the incubations were at 37°C since that’s where E. coli likes to grow. However, this transformation didn’t work and we got absolutely no colonies.  I tried it again with the same results, unfortunately.  There just didn’t seem to be enough of the DNA to get put into the plasmid. It might seem strange we were trying to get the plasmid into E. coli, since we ultimately want to see how it acts in P. putida. We did this because we needed to make sure we actually had a plasmid because putting the plasmid into P. putida is more difficult than getting it into E. coli. But since we couldn’t even get it into E. coli, there was no hope for Pseudomonas.
 

Then, Kati remembered that she had a successfully mutagenized pKG207 plasmid from February that we could use to transform into P. putida. That’s exactly what we did. First, we transformed it into the wild-type, which worked! However, because wild-type would have the oxidation gene on their chromosome as well as on the plasmid, it would be difficult to deal with the interference and analyze the oxidation phenotype. So, the next transformation we did into KG127, the non-oxidizing mutant, which also worked very well. The KG127 cells were made competent by me the way I was explaining earlier, whereas the wild-type were pre-competenized (not actually a word) cells. There have been a handful of promising colonies that look to be oxidizing a lot, so we are going to be screening those soon and possibly accomplishing, or working towards accomplishing, the original project Kati set up for her summer student. Yay!
 

Another test we continued that became slightly tedious this week was the membrane-active agents test. We were testing using sterile filter discs soaked in 10 µL of various dilutions of ethanol, EDTA, SDS, and hydrogen peroxide placed on lawns of bacteria. However, we did 4 discs on each plate and we plated a plus and minus manganese lawn on a minus manganese plate and a plus manganese plate. So for each agent there were four plates with four filter discs each. That meant placing a total of 64 sterile filter discs and carefully pipeting 10 µL of liquids which I had to make up dilutions of. It took a lot of time to set up and to be careful throughout the entire set-up. I actually enjoyed setting up this particular experiment though because it became a sort of mindless task and I just listened to music while putting it all together. The results from this test were not very drastic, but Kati and I could see faint halos of oxidation around the discs of SDS and EDTA, insinuating that oxidation might be a stress response to the membrane-agent, or that whatever interaction it had with the membrane caused an increase in manganese oxidation. However, since the ring was so faint it is difficult to draw any conclusion from these tests. We are still trying to come up with a way to make it more quantifiable.
 

This week I have also finished up testing for oxidation as a protection against UV radiation. I did a few final tests with the Stratalinker at various energy settings. I ended up getting two plates at 10,000 µJ of energy where the oxidizing clearly survived and the non-oxidizing clearly was dead. These results are pretty convincing and are what I’m using in my presentation and are attached to this blog entry.

On Friday, I attended two presentations. The first was given by Jose Lemos, who was visiting from the University of Rochester. His presentation was very interesting; it was about Streptococcus mutans, a strain of oral bacteria, and their stress responses in the mouth. I enjoyed it because I learned a lot about this strain of bacteria in my microbiology class and actually gave a presentation touching on it a little bit in my honors chemistry class. It was also relevant to my research because it dealt with stress regulators and responses, just like how Kati and I are looking at oxidation in P. putida. Two months ago a seminar such as this one would have been overwhelming, but I really enjoyed and understood it. I have really learned a lot here. The second presentation was given by a grad student from PSU and her life and the various jobs she has had throughout it. She spent a lot of time emphasizing how variable jobs can be and that the prestige does not necessarily correlate to enjoyment or satisfaction or money. I have found that my job, which is really an internship, has not always been the most enjoyable, but is an amazing experience nonetheless.
 

Writing this blog entry took a lot of time and was another busy-bee job for me. Actually writing out and organizing everything I have done this week has really solidified my understanding of the experiments. Now that I understand the procedures, I don’t feel like I’m wasting time and I really feel accomplished when the experiments turn out well. Even the long and tedious set-ups and protocols can be enjoyable for me, as long as I keep busy. Busy work isn’t always such a bad thing. Keeping busy writing this blog, preparing my final presentation, and writing my final paper may seem stressful, but it’s really been a lot of fun, and I am feeling very accomplished.