The Sixth Week – Remembering the Big picture

When we are doing our experiments, it’s easy to get sucked into the very minute details and forget the big picture. We are testing one type of environmental stress on a specific strain of Pseudomonas that is growing on a specific type of media under specific temperature conditions for a specific amount of time. And the amount of bacteria is so tiny; 100μL aliquots of liquid at a time. Such small amounts as well as the need to keep everything very sterile and test several seemingly small variables makes the lab work very tedious and time-consuming. But it is important to try to keep things in perspective and remember why we are actually here researching this problem.

 

So, why are we really researching manganese oxidation performed by Pseudomonas putida GB1? I’ve been working specifically on why P. putida GB1 oxidizes manganese, but the research being performed by the Tebo lab encompasses the entire process of manganese oxidation, not only why but how and what forms of manganese and several other things. But why are we interested in this specifically? The cycle on Manganese in the environment is important because it has strong effects on biogeochemical cycles. The oxides have sorptive qualities that allow them to control the availability of many toxic and essential elements. By understanding how bacteria catalyze the manganese oxidation and effect the entire cycle we will better understand how our environment works and how we might be able to use these bacteria to our advantage to do things like clean up toxic metals. So that’s the big picture, that’s really why we’re doing this research. 

 

But now I’ll tell you about all the seemingly small experiments I have been working on to work towards these big ideas.

 

This week I continued working on how GB1 holds up to UV light by testing it against UVA and UVB light wavelengths. We had been having trouble with getting accurate and repeatable results, so we decided I should wear glasses and look at the lights we are exposing the bacteria to make sure they were working correctly. It turns out they weren’t, and many of the lights were flickering and giving uneven amounts of light. So I repeated the experiment trying to avoid the flickering lights and expose the bacteria to a more even amount of radiation, but we’re still having technical difficulties. It seems pretty obvious that the oxides do protect against the other forms of UV, we just haven’t gotten pretty enough plates for pretty pictures yet. I’ll continue working on that.

 

We have also begun testing GB1 against different membrane-active agents including: SDS (a detergent), EDTA, ethanol , water, and hydrogen peroxide (H₂O₂). We started out adding a variety of dilutions of each of the agents to an oxidizing and non-oxidizing sample of GB1 in a 96 well plate and taking the optical density measurement to see what grew or didn’t grow as well. The results weren’t very definitive and we think it might be because they couldn’t grow very well in the plates while not being shaken. So we took a step back and made cultures in tubes of stronger dilutions (we based our dilutions off of minimum inhibitory concentrations) with both non-oxidizing and oxidizing bacteria to see if the oxidizing bacteria can hold up better. All of these dilutions for this experiment are a great example of the time-consuming work that may easily get your mind to stray from the big picture. I made 4 dilutions of 4 different agents for oxidizing and non-oxidizing bacteria the first day. I made 5 dilutions of 5 different agents for oxidizing and non-oxidizing bacteria the second day. And then I made 3 dilutions of 5 membrane-active agents for oxidizing and non-oxidizing bacteria a third day. That’s over one hundred different cultures each with a unique amount of each ingredient that had to be calculated and then added to different tubes or wells, which is why it is so time-consuming and easy to get lost. But it is valuable lab experience that I am lucky to have. To analyze these cultures we have been measuring the optical density to see what grew and what didn’t… however the numbers have been less than helpful. There doesn’t seem to be any noticeable difference in survival of oxidizing and non-oxidizing, and some of the agents that should kill the bacteria seem to help it grow more. So who knows. Another things we tried in the oxidizing mixtures was adding LBB and then taking the optical density to see how blue each culture was and to see if oxidation was induced under stress. However, again, these numbers were less than helpful.

 

We also began the project that was originally planned for me this summer, which is to amplify the MnxR gene that encodes for the protein that is responsible (or at least partially responsible we think) for oxidation of manganese in P. putida GB1. First, Kati explained the process of PCR(polymerase chain reaction) to me. It is the amplification of a specific chunk of DNA and replicating it a lot. We did this for the part of DNA that encodes our protein. Actually, we did it twice. The second time we used different enzymes to get a bigger chunk of DNA so we could later give it “sticky ends” when we perform the digest. We did this because we need to get this piece of DNA into our bacteria, but they don’t like just chunks of DNA, they like circles of DNA, which are more scientifically called plasmids. To insert the chunk of DNA into a plasmid that P. putida likes it needs to have sticky ends that will allow it to be inserted in the right orientation, without the sticky ends it can get inserted backwards and that would be a problem. To actually perform the PCR, we mixed together a very small amount (sometimes less than 1 μL) of the DNA template, DNA polymerase, dNTPs (what makes up DNA), primers, and a buffer to put into a PCR machine that automatically cycles through temperatures where DNA denatures and then sticks together and replicates for the correct amounts of time. Actually it is a denature, anneal, and then extension cycle. The PCR machine is very handy because doing the cycle of 95°C for 20 seconds,  60°C for 20 seconds, and 72°C for 45 seconds 30 times over by hand would be extremely tedious. And would definitely help me loose site of the big picture.

 

When the PCR is complete we need to check the results and make sure the PCR was successful and we do that by running the resulting DNA through a gel. It sounds odd, but that’s really exactly what we do. We first poured a gel mold and let it harden. The gel has wells at the top to insert the DNA into. The gel is put into liquid and then our small amount of DNA mixture is mixed with a dye and some glycerol to make it heavier so it actually drops into the well. Then we send electricity through the gel and the DNA runs down leaving bands of dye depending on its size. We then photograph the gel using UV light to illuminate the bands. We actually used the light box I’ve been using to kill bacteria for the photographing, its original purpose. We put in a standard “ladder” along with our own PCR solution to compare the lengths and make sure the DNA we have is the length of the DNA sequence we wished to amplify. And it was, however there were a few other lighter bands of different lengths possibly from the plasmids we had in the mixture too. So next week we will continue with the process to randomly mutagenize (if that is even a word) the DNA and get it into P. putida.

 

This week I also met with Brad Tebo. I went in to see him on Thursday with Kati for about 15 minutes to talk to him about how my internship is going and about my results. I was nervous, but once we were talking to him I felt very comfortable. He seems genuinely interested in my research and results, and he is really good and thinking about the big picture. Talking to him got me excited about what I was doing again and I actually felt like I was making progress.

 

On Friday, all the interns took a trip to OHSU school a medicine for some tours and mostly information about grad school. The campus was beautiful. First, three grad students gave us a tour of their labs and had us ask them any questions we had about anything. It wasn’t really relevant to my future track because I am an engineering student and would not come to OHSU, but it was really cool to talk to them about their lives and their research. Going through their labs was also fun because they looked pretty much exactly like the lab I work in! It made me think about how this experience I’m getting is so wonderful and could really help me other places. We also had several other people talk to us about grad school, about public health, and just research in general. The trip wasn’t helpful for me specifically because it is a school of medicine, but it did get me thinking about my future again. This summer has given me a taste of research and allowed me to realize that this isn’t exactly where I want to go with my schooling, but I am enjoying the opportunity and unique experience. I do think grad school is something I will pursue in the future, but only for a masters degree, and hopefully a future employer will pay for me! I love the idea of having more individualized and self-motivated learning like I keep hearing about at a graduate education level.

 

The tour to OHSU and talking to Brad really got me thinking about the big picture again. I may be encompassed in the small and tedious lab work, but I can also remember why I’m here.  I value this experience where I am adding to a larger body of knowledge and furthering myself and my education. I can look at the big picture of science and see how the research I’m doing is adding to that, but I can also look towards the future and see how this experience will help shape me and gives me opportunity to further my education and work towards a career that I am passionate about.