The Seventh Week – Solving Problems

Throughout this internship I’ve felt very out of my element, as I’m sure I’ve made fairly clear throughout my blogs.  I’m very new to laboratory work, biology, microbiology, and especially genomics.  But I have still found some connections from what I’m doing here to my chosen career path in environmental engineering.  Science and engineering are not normally closely linked, but there are some main similarities that permeate both professions.  The one that I’ve experienced the most is problem-solving.

I define an engineer as someone who solves problems.  Many people label engineers as people who build things, but truly engineers solve our world’s problems using many tools like math and physics.  Scientists do the same thing; they solve problems.  Scientists have a different focus and the problems they aim to solve are more knowledge-based.  Research is all working to enrich the larger body of knowledge that is science.  They are solving the world’s mysteries and in doing so, run into many problems to solve along the way.

One of the most frequent problems a scientist runs into is how to get funded for their research.  This may sound superficial, but it is a huge consideration.  Most of what I’ve been working on this summer was to supplement a grant proposal to try to fund the Tebo lab’s research.  This week, on Friday, we had a four hour seminar just about how to write a good grant proposal that will get funded.  It was a lot of information a bit early for an undergraduate like me who doesn’t really plan to move on to get her phd.  However, it reminded me that one of the biggest hurdles in doing research, or doing anything really, is getting the money to support your project.  Once the problem of funding has been solved, many more lie ahead.

Problems arise in every single experiment that must be reconsidered.  My UV tests have been a good example of this.  I was having problems getting good results from the light box with different wavelengths and the lines were fuzzy where I was trying to block the UV for different lengths of time.  This caused our pictures to  be not so pretty, and pretty pictures are important to articulate the point proven by the experiment.  But this week, after some modifications, I finally got my pretty pictures!  The very obvious difference between protected and not protected has been captured.  Another thing we’ve been trying to solve is how to quantify the protection from UV.  Kati said that many other experiments list how much energy they exposed the bacteria to in joule measurements and have fancy machines where they can adjust the wavelength of their light.  Although we don’t have one of those, we do have a UV crosslinker.  It is supposed to be used do get DNA to attach to polymers or something like that, but really it’s just a UVC light box, with time and energy settings.  So we have changed to measuring the amount of radiation the bacteria has been exposed to in μJ instead of seconds.  By figuring out what amount of energy the oxidizing and non-oxidizing bacteria can withstand and comparing them, we should be able to quantify the protective effect of the manganese oxides.

Another test of our problem-solving skills that we still haven’t overcome is the membrane-active agents test.  We began them in a 96 well plate and tried to take optical density measurements to quantify how many died and didn’t and if the oxidizing strains had a higher survival rate.  However, these results were altogether unhelpful.  We moved back to tubes to try to grow them while shaking and saw a visible difference in growth, but when we tried to quantify it, we had problems.  Our idea was to use Coomassie Protein assay which turns blue when there is protein, so if there is more protein, the more blue the solution will be.  We figured we could do a quick test of this and add it to cells which would lyse with vortexing since the assay is so acidic and would turn the cultures different shades of blue.  However, all the cultures turned very blue, seemingly no difference even though before adding the protein assay it was obvious there were far less growth in tubes with, say, 3% hydrogen peroxide.  We have again decided to take a different approach.  The liquid cultures have always caused us troubles with clumping and now with the protein assay, so we have gone back to plates.  We have plated lawns of bacteria, oxidizing and not, and added sterile filter discs soaked in various dilutions of the membrane-active agents.  Next week, we will see if there is a clearing of dying bacteria and how  big the clearing is for different agents.  Ideally, the oxidizing plate would have a smaller clearing than the non-oxidizing bacteria if the oxides really protect the bacteria.

The other project we’ve been working on is with the PCR product and using transformation and ligation to get the product into a plasmid that can eventually be taken up by P. putida.  After the PCR has been complete, the product must first be digested using the sphI enzyme, which we ordered and just got in Friday afternoon.  Since we didn’t want to wait until Friday to try it, we borrowed some restriction enzyme from the lab across the hallway.  Once the product was digested we ran the entire product through a gel and then cut out the band of DNA we wanted.  We did the same digestion with the pUCP22 plasmid too.  Once we had the cut out pieces of gel we used a purifying kit to cleanse and then store it in the -20°C freezer.  I got my own box with my name on it with my two pieces of DNA, which I think is kind of cool.   So now we have little pieces of DNA, with “sticky ends” due to the enzymes we used to cut the ends and we tried to use ligation and transformation to get the bit of DNA stuck into a plasmid and into P. putida.  We let them grow at 30°C, and then replica-plated them to lept plus gentamicin plates to see if any of the colonies oxidize a lot.  Since that is what we want to end up with ultimately.  I still have difficulty understanding all of this DNA PCR, digestion, ligation, and transformation stuff… I can’t really wrap my head around it all.  But it’s a process and I’m learning it over time.

The knowledge and experiences I’ve acquired since my time here in the lab will definitely help me in my future as an engineering student.  Although I will not likely be dealing with a lot of the same scientific material that I have encountered here, I have been able to see that skills I’ve used in my internship will also come in handy for my future career path.  The need to solve problems and think of creative solutions every day are things that will help me throughout my life, and especially as an engineer.