Week 1- Grow bacteria, grow!

Hello there! My name is Logan Smesrud, and I am an environmental engineering student at Oregon State University. I am a summer intern at CMOP through the Johnson Fellowship program, organized through my university. The Johnsons are alums of my engineering department and started this program specifically for chemical, biological, and environmental engineering students who have just finished their first year. Their mission is to give young undergraduates a taste of research and real work experience at the beginning of our undergraduate career. They are an incredibly generous, and kind couple. I am so honored to be a Johnson Intern this summer. Thank you, Pete and Rosalie Johnson, and thank you CMOP!

As an environmental engineering student, I don't have much of a focus on genetics or biology, which is the focus of my project here at CMOP. This means that I have to do a little bit extra preliminary research to remind myself of key terms for talking about bacteria and cell growth. I am excited that by the end of this internship I will have a much stronger grasp on genetics and bacteria growth, as well as general lab and research techniques. I'm super excited to get started!

To start off my first day, Vanessa Green gave me and all of the other interns an introduction to CMOP and OHSU. She gave us all calendars which include fun excursions that she encourages us to go on with her and other people in the lab. After taking our pictures for the ID cards, Vanessa kindly took us out to lunch! Then, she dropped us off in our labs and I met my mentor, Dr. Kati Geszvain. She is incredibly organized, and I really like that. She had already printed off a few her papers so that I could get a sense of the background of her project, which she has been working on for five years now! She showed me around the lab and pointed out instruments and materials that I will need frequently. I’m excited to get started!

On Tuesday I made media! Dr. Kati had written up recipe lists for me to use. It was a lot of pouring, measuring, and autoclaving. I appreciated that Kati watched me do all of these steps to make sure I didn’t mess them up. After the media was ready, I poured them into plates. It turns out that “pouring plates” is quite an art, and one that must be mastered with experience. Kati showed me her technique and then had me do the rest. I spilled one plate today… which was loud and embarrassing. Luckily Kati pointed out that I had poured 21 plates already and we only needed 20, phew! Kati printed out some more papers for me to read when there’s downtime between experiments.

On Wednesday morning we took the E.coli and pseudomonas putida plates out of the incubator, and my bacteria grew! Woohoo! Each of the two E.coli strains we grew lacked a gene needed for manganese oxidation, but contains a plasmid that we will want to transpose into the p.putida when we conjugate the bacteria. This week is kind of a “get things ready week” because we had to make the media, grow the bacteria, and prepare a few other things before we can delve into much more. Luckily I LOVE the little pipetteman tool. It’s so snazzy how you put the tiniest little tip on it, you tell it exactly how much you want it to suck up, and then when you’re done there’s a button that pops the tip right off! It’s pretty fun! I also prepared two plates, each with six sectors drawn on, and streaked each area with my bacteria that was lacking different genes that I had prepared the night before. I put one of the plates at room temperature and the other at 30 degrees C. “Streaking” the plate is quite an art, and one that my mentor preforms so gracefully. Since so many things need to stay sterile and I can only touch certain spots of containers, I feel like I am getting used to the size of my own hands. On Wednesday I also had email/blog and safety orientation sessions, which are required for new OHSU employees.

Thursday was a good day. It really feels like concepts are clicking now. As Kati was explaining something to me today, with helpful diagrams and all of that good stuff, I asked her a hypothetical “what if” question and she responded with an excited clap of the hands and said “Exactly! That’s what your project is!” Kati had drawn a flowchart of genes, and that’s when it all made sense: if we delete the initial regulator gene that controls the expression of the following two oxidizing genes, but also delete the unknown negative regulator that is on one of the two oxidizing genes, then manganese oxidation will still be expressed with the deletion of the regulator gene. I had a hard time wrapping my head around what exactly my project was until today, when the light bulb went off.  I made a few sub-culture tubes so that we can bring the stationary-phase bacteria back into their exponential phase to grow more. I went to one of the graduate student’s thesis defense today. Although I didn’t understand everything she was talking about, it was a good experience to see the format and presentation of a thesis and its defense. Later that day, we started a fun little side experiment where we put the putida cells, a manganese chloride solution, and various carbon sources in 4 different tubes. We want to see if perhaps the bacteria oxidizes manganese as a way to help it break down complex carbon sources to a size that it can consume it as food.

On Friday I continued the conjugation of my bacteria by transferring them from the one plate that they had been growing on onto multiple other plates to continue their growth. I also continued the carbon source pilot experiment by extracting the bacteria and using an LBB indicator that turns cobalt blue in the presence of manganese oxides. Indeed, what we were seeing was manganese oxidation. To quantify the level of manganese oxidation with the various carbon sources, we used a spectrometer to compare absorbencies of the blue colors, and of the cells alone. This way we wouldn’t just assume that one tube did a better job oxidizing manganese when really it might just have more protein in the tube. After all the science, we went to a kickball and ice cream social with other people in the lab. What a fun way to end my first week here!