Grad Diary 7/15/2009

So, today I had a lab meeting with Dr. Fraser. It was my first formal meeting with him since I've started in his lab almost a month ago. Iryna was with me too. It went pretty well, although in the beginning I had my doubts. His opening question was, "pretend I'm one of your classmates and explain to me what you're doing." This took me completely by surprise because I was expecting to give him a status update, not a mini talk. While I knew the basics of what I was doing, I didn't feel prepared to talk about all of the biology involved in my project - I would need to review some material before that, so I was a little flummoxed. It was also weird trying to give this little talk to the mastermind behind the whole project to begin with.

After stammering my way over some of the introductory stuff I caught my groove once I started talking about my projects, and things went smoothly after that. Predictably, Dr. Fraser said I should give more details on the background, but he was impressed with my explanation. He had specifically given me no warning before hand to see how I would do...apparently he does this a lot, especially to newbies.

He probed me a bit during my explanations to make sure I understood not only what I was doing but why. This wasn't a big problem because I generally make a commitment to knowing why things are done the way they are done. It's very easy, especially in microbiology, to get sucked into running protocols and not understanding why each step is needed or what the underlying scientific principles are.

I did run into one small crisis when I was talking about bacterial transformation. This is a process where you introduce novel genes into bacteria, which are very good at picking up foreign DNA and incorporating them into their genomes. While this can be a pain when bacteria share antibiotic resistance, it also makes it easy for us to engineer them to make things we want, like human insulin. Anyway, I was explaining how I was constructing my target plasmid by isolating my desired insert using restriction endonucleases and ligating them into a vector plasmid. One important step is dephosphorylating the vector plasmid after you've cut it open. I kind of brushed over this and Dr. Fraser asked me why we would dephosphorylate the vector and not the insert. It had been a while since I had reviewed the actual science behind restriction endonucleases and plasmid construction, and I was stumped for a moment.

Before letting myself panic, I calmly started to reason out loud why this would be the case. "Well, let's see. You need a phosphorylated 3' end if you're going to add a nucleotide since the polymerase doesn't add one, and you also need one if you're going to do ligation..." and as I said this in my mind I saw an endonuclease cut up my vector, making a nice open space for my insert. On the opposing 3' ends were bright yellow phosphate groups (phosphates are always yellow in biochem texts). I then pictured a DNA ligase coming in and before my insert was able to move in place, it neatly sealed up my vector back to its initial state. This all took probably a second and suddenly I knew the answer.

"Oh, of course. It's to keep the vector from re-ligating and excluding my insert."

"Very good!"

I felt a mixture of pride (for thinking quickly on my feet and being able to reason it out) and stupidity (for not knowing this sooner). All in all, Dr. Fraser was happy with my progress and impressed with my level of understanding, which made me feel pretty good.

Lessons of the day: 1) Make sure you understand the science behind everything you do. 2) If posed a question you don't know the answer to immediately, calmly reason it out using what you already know.