The Third Week – The Challenges and Successes of Learning

Time has been going by so fast; I can’t believe how long I’ve already been here or how much I’ve learned. I suppose that’s what this entire internship is about: learning. And this week has been full of it.

 

I believe the best learning comes from mistakes and overcoming challenges. I know, because of my lack of experience, that I will make many mistakes, but I also know that these mistakes are what help me learn. Science is also all about making mistakes, just trying something and seeing what happens… and altering your course of action from there.

 

This week actually started on Sunday for me. My mentor and I came in to look at our UV experiment from last week that we performed on several different mutant strains of GB1, and it was puzzling. Our results seem inconsistent with previous results because some non-oxidizing survived very well and some oxidizing strains showed little to no difference in its survival of UV while oxidizing and not oxidizing. And the problem-solving began. We decided to scale back to our original strain plus two mutants: KG 127 – a non-oxidizer and KG 139 – a super-oxidizer.

On Monday, I inoculated the GB1, KG 127, and KG 139 cultures in the afternoon like I would usually do, along with quite a few other cultures. Tuesday morning, I was surprised; all the cultures grown with manganese chloride were oxidizing, even KG 127 – the non-oxidizing strain. The most logical explanation for this was that I made a mistake during the inoculation. I mentioned earlier that I know I will make mistakes, but it was disappointing and a little frustrating. However, these sorts of things are going to happen throughout my internship, and I know I can learn from them. This mistake delayed our UV experiment a little bit because we needed different cultures that I inoculated Tuesday afternoon. Those cultures turned out much better, so maybe I did learn from my mistake.

 

This delay also allowed me to think a little bit. We were having some discrepancies in our data because, although we were plating 100 μL of culture on each plate, we couldn’t be sure that each plate contained the same amount of bacteria because of clumping during growth in the liquid. I was trying to think of a way around this, to make sure we are testing the same amount of bacteria at different lengths of exposure. Since when we spread the bacteria on each plate, it gets pretty evenly distributed, if we performed the UV exposure time scale on each individual plate, the concentration of bacteria for the area of the plate would be more even and create less uncertainty. When I suggested this to Kati, she said that it sounded good and we should do it. Although it was a very small change, I feel somewhat accomplished for coming up with a solution; a small success.

 

The UV experiments have been the most successful and encouraging experiments we’ve done. Today, we performed our 9^th test, this time repeating the previous test with wild-type GB1, KG 127 and KG 139 over a time course, but also testing some wild-type GB1 that had been treated with ascorbic acid to dissolve the manganese oxides. This will show us if the bacteria lose their resistance to UV when the manganese oxides are removed. I’m very happy about the UV tests we’ve been doing and excited to see a result for this new one too. Hopefully we’ll make a figure out of all our pretty pictures to put in the grant that is sufficiently convincing.

 

We also looked to see if the bacteria could utilize humic substances or hickory tannin (both being sources of carbon) for energy or how the addition might affect oxidation. At the beginning of the week, we looked at the effect in lept liquid cultures, and it appeared that they increased oxidation with the added carbon sources. So we’ve put them in Hepes, which has little in the way of food for the bacteria, to see what would happen if the extra carbon source was its only source for nutrients. However, these results don’t seem very exciting. After three days of growing, there is still not very much happening in those cultures.

 

Another test we began this week is heat shock. We submerged the cultures for 1 and 3 hours at room temperature, 37°C, 42°C, 55°C, and 70°C. We then plated a dilution of these cultures to look at how many bacteria survived. From this test we determined that Pseudomonas putida definitely cannot survive 55°C and 70°C temperatures. However, the number of bacteria on each plate was inconsistent again, making it difficult to analyze the results. The amount of growth on the plates at 37°C and 42°C contradicted each another; at one temperature the oxidizing bacteria grew more and at the other temperature the non-oxidizing grew more. I believe this is again because of the inability for us to make sure we are plating the same number of bacteria. We also didn’t get individual colonies, just lots of growth together, making it even more difficult to quantify our results.

 

We also experimented with a conjugation to help out someone else with their work. I do not fully understand the process of conjugation, because of my lack of background in biology, so this required Kati to explain a lot of things and give me some materials to read. I sort of understand it now, but the steps in the lab are still very cumbersome and confusing. There is a lot of “hurry up and wait” (what Kati says) involved in the process.

We are trying to get a plasmid (circular DNA) inserted into Psuedomonas putida GB1’s genome to disrupt what we think is the sequence responsible for making the protein that plays a role manganese oxidation. We did this by combining E. coli that has the plasmid, a helper strain, and Psuedomonas putida on a plate and the helper strain helps the plasmid get inserted. The plasmid also gives the bacteria antibiotic resistance, so we will know if it worked if the bacteria survives on a plate with that antibiotic in it. So we hope that it all works by heat shocking them when treated with calcium chloride (a mystery why that works) and mixing them together and letting them grow. I don’t totally understand it, and I don’t think I was very successful in my first attempt, but we’re trying again next week.

 

All week I’ve been learning. I’ve learned from my own simple mistakes in the lab. I’ve learned a lot from my mentor Kati and also through reading many materials she’s given me. Kati and I have both learned from our experiments, and using our knowledge, as well as that of those around us, we are coming up with more ways to torment Pseudomonas putida GB1. Science is about learning. There are always challenges to be overcome and always new problems, but there are also successes, however small, that make it all worth it. That’s what I think learning, and this experience, is all about.