A Blog actually filled with relevant content!?
So yeah. I guess 24 hours somehow turned into 408 hours in the blink of an eye. Funny how that works. But as I sit here waiting for all of the data I have accrued over the past two months to compile itself onto my lovely, portable desktop, I felt that the world deserved some minor glimpse of my research, since I have done a relatively poor job of explaining it thus far.
With a grand total of 7 days remaing in the program, (one of which will be spent eating and displaying our research before anyone interested) I am finally beginning to realize how little I have done in progressing toward anything remotely close to a conclusion. Simultaneously I am discovering just how much I have learned, about science, about research in a developmental biology lab, about Drosophila, about academia, and about my own potential as a scientist. Instead of being lost in a sea of possible careers, fascinating subjects, and daunting years of schooling, I am now almost able to precieve the mainland through the curtain of dawn's receding mist. That being said, I still cannot describe my future, or rather the future I want for myself, but find myself thirteen steps closer to this achievement.
So, the past 7 weeks have been spent working with dorsal closure, as previously mentioned and explained. The past 4 weeks I have been doing experiments with Drosophila embryos expressing trpa1RNAi and rpkRNAi genes, each knocking out their respective mechanosensing proteins, transient receptor potential A1 and ripped pocket. Our hypothesis was that these embryos would completely fail to complete dorsal closure or at the very least progress far slower than wild type embryos. Through about 100 hours total spent imaging and analyzing these embryos I have discovered that yes, the embryos DO close slower and, despite the rare fluke, not only slower but much MUCH slower. It was definitely a satisfying result to receive, because it means that these two proteins are pivotal in dorsal closure. Additionally, and this is actually more interesting, although many of the embryos were able to complete closure, the often died in the subsequent stages of embryogenesis without these proteins. Ginger, my mentor, has observed a distinct failure in head development and this could definitely result in the death of the little guys. Cool, right? Whatever these proteins are doing, it is not just mediating tension in dorsal closure, but perhaps mediating and directing intercellular signaling throughout the organism. I guess I wish that the embryos had just exploded from too much rogue tension, but you win some and you lose some, right? ; )
I have has begun to develop some side hypothesis from pure observation and instinct, the former of which I am not good at and the latter of which I have not really developed yet, thus I feel like they are purely born out of a natural predilection for seeing extreme possibilities in less than extreme circumstances. So those will have to wait to be pursued and presented at a later date.
My other project, the infamous CLONING PROJECT, has come to a sudden violent stop. Kind of. So the goal of this project was, as you might remember me hinting at, to create a transgenic fly expressing a GFP tagged antibody for phosphorylated tyrosine. This chemically modified amino acid is often found in proteins involved in signal transduction pathways, and thus seems like JUST the thing to be marking in our current course of research. Additionally, although the construct has been synthesized and expressed in other organisms, it has (to my knowledge) not been expressed in Drosophila melanogaster, and thus would be a great contribution to the fly community.
So the specifics of the cloning, the infinite number of recombination reactions and overnight liquid cultures, picking plates and then re-plating bugs, is far from exciting, but until yesterday the results had compensated. We successfully recombined the py20 gene (the phosphorylated tyrosine antibody gene) into a plasmid containing a 6x (6 amino acid) HIS tag to help identify the expression of the antibody in a western blot if necessary. We then transferred this whole construct into an expression clone and have now induced expression over the course of the past 2 days. And have seen very little evidence of a band indicating presence of the antibody. Bummer, right? all that work for nothing. Of course it could easily be due to the conditions of induction, the antibody could be toxic, the auto-induction media could be faulty, the list of possible errors goes on an on. And that is the hard part we now must commit to. Finding/troubleshooting, isolating, and repairing the mistake in induction. YAY for millions of variables shifting constantly! But life goes on, and it is always important to keep the ultimate goal in mind because it is so marvelously cool.
On the fun side of things, I saw watchmen recently and, while not being phenomenal, it surpassed many of my expectations since all I had heard were terrible reviews. I would recommend it to anyone and everyone, just keep an open mind and try to recognize the underlying messages present throughout the film. Plus who doesn’t love to stare at a ripped, naked, floating blue guy for 2 and a half hours?
