Colin King, Weeks 2 and 3

Colin King, Week #3, The Smell of Water

As I approach the end of my third week at the Bissig Lab, I’m finally beginning to achieve a sense of familiarity and comfort with my surroundings and the people who work around me. Dr. Pankowicz has returned, and though I usually don’t see him till he arrives after lunch (but apparently stays till 2 am), he has been extremely friendly and done a great deal to help me understand things however I have trouble or ask him questions (which is pretty often).

Since my last entry, I have successfully synthesized the two gRNAs (guide RNAs) individually for each of the genes I was assigned to (Agt which is responsible for the creation of angiotensin II and DDC which regulates dopamine uptake). Since we want the Cas9 enzyme to effectively cut out one portion of the gene, from one exon to another, and each gRNA targets one exon in the gene, I now have arrived at the step of “Daisy Chaining” the two gRNAs together in one vector, so the Cas9 can attach to one exon and cut the gene until it arrives the next one. This entails a similar procedure to the synthesis of the initial gRNAs, beginning with an initial digest, purification, then ligation into the vector, and finally a transformation with (now with a different type) of e coli. However, since the insert is much larger, various complications arise that often require me to do things over and over and over again. I heard this joke the other day that PCR actually stood for “Pipette, Cry, Repeat”. Once my sequencing results come back and I confirm that I successfully created the vector with the Daisy Chained gRNAs, I can start a maxi-prep to amplify my product, which will subsequently be injected into a mouse.

The Nanodrop, used to quantify DNA

We determine whether or not we were successful at synthesizing the gRNA by sending the sample off for sequencing, and checking the results with the nucleotide code that we wrote on the computer and manually "inserted" into the gene to create the gRNA code.

Disappointingly, Dr. Bissig has informed me I will not personally be able to perform the hydrodynamic tail vein injection myself, as I am 17 and BCM has policies against minors handling live animals. However, maybe its for the best, as when I was talking to Nika the other day as she was doing the online mouse training, she exclaimed in language that I probably can’t write on this blog that the course was incredibly repetitive and time consuming.

This week has not been without its challenges though.  For some reason, a few days ago, I opened my vial of molecular grade water (which is something added to most of the reactions throughout various steps of the cloning procedure) and took a whiff, obviously expecting it to smell like water—nothing, odorless. I was taken aback when I realized it had a strange, sour smell. I panicked, realizing it must have been contaminated and that I had messed up every single step of the procedure, and would now have to start over again, sacrificing two entire weeks of work. I tentatively asked Sebastian, who was working on the bench next to me to smell the water himself and confirm my doom, and he too wrinkled his nose after taking a whiff, making my heart plummet from the disappointment that I had made such an idiotic mistake. I was about to head towards the freezer to retrieve my oligos and start over when Mercedes saw us smelling the tubes of water and asked us what the hell we were doing. When I told her, she laughed out loud, and quickly explained that it was the vials that smelled like that, and showed us an empty tube to smell in order to prove it. I will never forget the smell of water.

I’ve also started to work on synthesizing some gRNAs for Mercedes for two other genes. I’m beginning to think that the other lab members also trust me more in my capabilities for lab work. Often times now, I run column purifications for Nika or make and run gels for Sebastian. The other day, Dr. Pankowicz instructed me to perform 20 transformations, followed by 40 minipreps (which is a way to take the bacterial growth and elute the DNA for later use). I had done both before, but never had I done so many at once. me. I wish I could say I enjoyed the process of repeating the same procedure 40 times, but somewhere around the 35th time I was extracting the supernatant during one step of the mini prep, I had lost almost all of my initial determination and zeal. Pipette, Cry, Repeat indeed.Still I’m glad for the practice and oppurtunity to gain a sense of familiarity with the procedures. Going through an entire pipette tip box in less than 20 minutes is a personal record I probably won’t beat, unless Dr. Pankowicz wants me to do 30 transformations next time.

The only time I can claim to have lifted 20 plates at once

In the past week I’ve also been able to familiarize myself with the Texas Medical Center area. Although I usually bring my own lunch since I don’t like going all the way down to the first floor and walk to the cafeteria (which, admittedly, is pretty damn good; the cafeteria feeds the doctors from the Ben Taub Hospital as well, and I guess doctors like eating well), I’ve discovered that there are a huge range of food choices within a half mile radius. 

With just six weeks left, it's hard to believe that I’m already one third of the way done with my time here at the Bissig Lab