The Fourth Week – The Importance of Communication and Collaboration

Science is not something to be done in isolation. Some people (even a past intern as Vanessa has mentioned) have said they want to become scientists so they don’t have to talk to people, but that is so far from the truth. When done in collaboration, science is much more efficient. Throughout this internship I am experiencing this first-hand.

 

Every week, there is a lab meeting with everyone in the Tebo/Haygood lab. We either have a round table where everyone talks about the recent progress on their project or one person gives an in-detail presentation about their project. These meetings are important for input and communication about research. Not only do we learn about what other people in the lab are working on, but we can contribute ideas and ask questions to push them and help them along in their work. I think this is a vital step in the research here at CMOP and fun too. Another thing I like at the meetings is that they ask if anyone has any praise or appreciation to give to anyone for helping them out in any small way, a nice way to remind people to say thank you and help each other out. Kind of gives you that nice warm-fuzzy feeling.

 

This week, Kati and I did a lot of experimenting, which led to a few failures and some confusion, but also some successes.  A lot of our experimenting was aided by collaboration with others in the lab for ideas; and we came up with some very helpful solutions. The experiments we conducted involved heat shock, UV exposure, differing carbon sources, and conjugation/transformation, almost all aided in some way by others working in the lab.

 

At the beginning of this week, we took a look at our carbon source liquid cultures again, but there still wasn’t much going on. We took optical density measurements again to compare them to our original measurements. Optical density is just measuring how much light is absorbed when shone through the culture. These measurements didn’t seem to have changed very much and were also complicated by the bacteria clumping in the liquid. Obviously, we needed to try something else. We decided that clumping in the liquid was a problem we didn’t want to try to cope with, so we changed our testing method and instead made plates containing the different carbon sources. We then struck GB1 and its mutants onto the different plates to compare how they grow or oxidize differently in the presence of different carbon sources. We also didn’t want to stress the bacteria as much as before with the hepes, since it didn’t survive very well at all. We needed to do lept plates to see the oxidation and we needed to have three different types of carbon sources, along with other things required to live such as trace elements and case amino acids. And in plus and minus manganese forms. However, we didn’t want to make 20 of each kind of plate, because we have six different types of plates… and that’s a lot of plates to pour. So we ended up talking to people in the lab about it and Sung-Woo came up with the idea of making three flasks each with the different carbon source and pouring half of it, then adding manganese chloride half-way through, and pouring the other half. We thought it was a pretty good idea, so that’s what I did Tuesday. Made a lot of plates.   I then struck out 5 μL of wild-type, KG 139, and KG 127 that had been grown in LB cultures overnight (so as not to get clumpy) on each type of plate. However, these results have still been unclear. After repeating the test several times with varying strains of bacteria, the differing carbon sources don’t appear to have a definitive effect on growth or oxidation. Also, a problem we ran into is that the bacteria didn’t grow nearly at all on the plates without manganese chloride in the media. We thought that might be because they need a minimum amount to survive, so I calculated (and it was a lengthy, and slightly tricky, calculation) how much manganese chloride I would need to add to each plate and let dry to acquire the same concentrations as in the other plates. However, this method didn’t work… when the bacteria were grown on the plates without manganese originally, but added manganese afterwards, they still didn’t grow very much. Kati and I will have to play around with this some more, and maybe talk to some other people in the lab about manganese requirements of Pseudomonas putida GB1. We still have quite a few of those plates to play around with.

 

This week, we have also been trying transformation to continue to help John out with his project. However, it hasn’t been terribly successful. We tried to make “competent cells” two different ways; the first way we tried is a “quick and dirty” sort of method. Competent cells are cells that will take up DNA (a plasmid) through transformation. The plasmid we want the cells to take up encodes the interruption to the DNA sequence we want as well as antibiotic resistance. When we struck the bacteria that had supposedly been competent and given time to take up the DNA, out on plates with the antibiotic that the plasmid encodes resistance to, we had about 20 colonies. We screened them and ended up with 5 that could be Pseudomonas putida, but those colonies have been growing all week and just seem sickly. We tried a second way to make competent cells that is supposed to be more reliable. When we used these with the DNA we want to be taken up, there were absolutely no colonies, so the next time we did it, we did a control plasmid along with the one we are trying to get taken up. Neither worked. So, evidently, our cells just weren’t very competent. We will try a new approach to this next week.

 

Again, the experiment that has been going the best is the UV testing. We have also been getting a lot of help from people around the lab that is helping us test even further. Last week, on Friday, I made some plates with oxidizing Pseudomonas putida that had its oxides dissolved by ascorbic acid. We talked to John about how he uses ascorbic acid in his experiments, how to make it, and what sort of concentrations to use. They turned out well; the plates with the ascorbic acid showed little to no resistance against the UV light, supporting our hypothesis that the oxides provide protection.  We repeated the ascorbic acid test and the normal UV test with wild-type, KG 127 and KG 139 mutants but we also added another test.   Roberto has been making some manganese oxides and Kati thought it might be interesting to see if adding those would protect the bacteria or not. He was nice enough to let us take a small portion of the oxides he has worked so hard to make to add to our little tortured bacteria. We added oxides until the liquid looked about the same shade of brown as the oxidizing bacteria did; obviously a very scientific method to measure oxides. We weren’t sure what to expect from these tests because although the oxides were protecting the bacteria, we questioned if the manganese must be oxidized by the bacteria to create a sort of shield or if being in solution with them would be enough. They ended up protecting the bacteria against the UV radiation quite well, so we’re testing it again. I think that they might be kind of sticky and that’s why they clump together and maybe cling to the bacteria to protect them. We decided we would test adding manganese oxides to cultures grown in LB where the bacteria can’t oxidize naturally, and see if they are still protected. They weren’t. Roberto also suggested we test E. coli with the manganese oxides too, to see if the particles simly scatter the UV light to protect all bacteria, or if they only protect manganese oxidizing bacteria. We have a lot of data and ideas to think about from these tests, and it’s very rewarding to be testing so many different aspects of this new discovery.

 

The other thing we played around with again was heat shock. Our first test was fairly unclear as I explained in last week’s post. So, today, when I had a bit of extra time at the end of the day, I performed a little trial test. I put some wild-type culture that had been grown in lept overnight into a 55°C heat bath and took out 5μL aliquots at distinct time intervals to streak out on a plate and see how, over time, the bacteria holds up to high heat. We chose 55°C because in our last test they all died at that temperature after one hour, so we should have the full range from all living before heat shock to all dead after too much heat. I was kind of excited about this because it was my idea to take out little portions at a time to streak onto plates. It will also prevent me from having to pour a million more plates. Well, not totally, but it will at least reduce the number. The results were that after 1 minute, all the bacteria had died. Evidently, the bacteria don’t hold up to high heat for very long. I’m not sure how we will proceed with these tests, or how far, since manganese oxides haven’t shown any sign of protecting the bacteria from heat shock.

 

Unrelated to my research, this week all of us interns took a field trip to Bonneville Dam and also Multnomah falls. The tour was nice, and our hike at Multnomah falls was beautiful. It was fun to spend some time with other people going through some of the same experiences. And get out of the lab for a bit too. We learned about the dam and how it influences the life cycles of fish, which I find both fascinating and disturbing. It requires the collaboration of many people and organizations to keep the dam producing power while keeping the fish safe, especially since the Columbia river is shared by multiple states. It would never be able to work so efficiently without the amount of communication that goes on. 

 

Throughout this internship, and as I’ve highlighted in this past week’s events, collaboration is an important step in scientific research. It is the only way to get a fresh perspective on an experiment, and a great way to acquire some helpful tools. Kati and I have gotten ideas from several people in the lab for almost every one of our experiments; we have also gotten bacteria and some home-made manganese oxides. Collaborating on ideas in any field is important and science in no different.  You cannot work alone.