Another school year has culminated, and the core of my upper division biochemistry major course have been completed.  Some of the classes I took were way more rigorous than I ever expected (Even though I had been warned about the terrors of the upper division biochemistry lab, I only thought people were over-the-top complaining.).  A few courses seemed to pass by at a snail’s pace, but now I have finished my junior year of college and it feels great.  I think that doing research helped me make it through the school-work because I was able to learn about science in an interactive way outside of studying textbooks.  Working in a lab and being able to figure out things for yourself allows more freedom for creativity than regular classwork.  I have always been the type of person that learns better by doing things on my own and designing my own way to go about the process.  My faculty adviser worked perfectly with me because he started me off on my own project under graduate supervision.  Although I worked closely with my graduate mentor at the beginning of sophomore year in order to learn the different laboratory techniques, eventually by the end of the school year I was working nearly autonomously.  I always discuss my results with my mentor and adviser, but I was devising my own ways to solve problems when an experiment didn’t work out.  I was especially happy when I figured out a mismatch dimer test to discover why I was unable to form a self-programmable RNA cube.  From my experiment I was able to see that some of the eight pieces that were intended to make a cubic molecule were mispairing and the cubic structure was unable to form.  I found that discovering things on my own in the laboratory was more satisfying than getting a good grade on a test and the excitement I got from biochemistry research has helped me make it through the tough classes.

Now that I am done with the majority of my biochem school work I am going to start taking classes in Evolution and Ecology in pursuit of a double major.  I am hoping that my future research will incorporate biochemistry and evolution….I will keep posting on my progress.  Happy summertime!

Finally I am sitting down to write about my research work this quarter.  Unfortunately I was unexposed to the rays of sunshine in Santa Barbara this spring because all of my time was consumed by studying, research projects, and organizational work with WiSE (Women in Science and Engineering).  My studying mainly involved writing one or two 40 page lab reports a week for an upper division biophysical analytical lab course that included several long in class experiments followed by days of computer programming to fit experimental data with Mathematica and protein amino acids into electron density maps with Coot.  Although I have learned an immense amount from this lab course, including how to create cool three-dimensional pictures of enzymes, the class left me with little spare time to study for other classes.  My research work was also put on the back burner during some weeks when I was scrambling to finish lab reports in the midst of midterms.  However, I continued doing my right angle motif characterization and it will not take me too long to discuss the results I obtained from the first few weeks of the quarter.  I only radioactively labeled two molecules because I wanted to focus on getting good gel electrophoresis results for a few molecules before moving on to other molecules, rather than trying to balance multiple experiments at a time.  Thus the first molecules that I labeled were two different right angle motifs.  I was testing the stability of the right angle structures by their binding strength to a probe molecule.  The more strongly the molecule binds the probe, the less rigid and more floppy the right angle because binding to the probe involves the right angle straightening out (ie, the 90 degree bend changes to a 180 degree line).  Unfortunately, the gel electrophoresis results I obtained were not the best quality; although I could see the titration curve of a probe-right-angle molecule complex forming at high concentrations of the probe and right angle.  Anyways, I am most likely going to have to re-label my molecules with radioactive phosphate and re-do the experiments for better results, but it should be more easy to deal with this summer when I do not have to worry about the hardest class I have ever experienced anymore.  Even though there were several times I thought I was having a severe mental breakdown when a misplaced comma would destroy my entire Matematica program, and I often regretted the amount of time my biochemistry lab class was taking away from me doing research, I am glad that I had the opportunity to go to a school where the biochemistry lab covered such a great extent of material and experimental methods.  I can now say that after three quarters of upper division biochemistry lab I understand a wide range of experimental techniques, from mass spectrometry to x-ray crystallography, to NMR, and how each of the techniques can be applied specifically to biochemistry.  And on top of that I can say that I know how to navigate a number of computational databases for biochemical experimental analysis, and I can definitely handle Mathematica.  So I its a good thing I spent a ton of time studying all the experimental methods employed by biochemists because I never know if any of these techniques may or may not be important to me in my future research, and I have also been introduced to methodologies that I find pretty cool and have sparked my interest in working with them in the future.  That being said I am quite happy that I now have all of that knowledge and can leave the stress of the class behind me.

In my last blog I talked about the intensity of seeing my polyacrylamide gel electrophoresis results since I have to wait overnight before I can scan the phosphor screen which has absorbed the spots where radioactive material was on the gel.  I was able to visualize the results of my gels from the first set of mutant experiments that I ran and the results were somewhat mixed.  I tested the fiber and square motifs along with two different forms of the fiber motif.  We expected that the fiber dimers would conglomerate together forming long molecules or “fibers” while the square dimer system could form different species in multiples of two.  The square motif looks like a corner to a cube.  Two of the sides to the square motif have kissing loops that are complementary to kissing loops on other square motifs.  Hence we have square A that has two kissing loops complementary to two kissing loops on the ends of the square B motif.  When the two pairs of complementary kissing loops connect they act as velcro sticking the two motifs together to form a table-like structure that could serve as the base of a cube for further RNA architectonics construction.  The square A-B system can form dimers like the table-like structure described or higher ordered species such as tetramers, however each species will be even-numbered because square A, containing A kissing loops, are only complementary to square B’s containing B kissing loops; they cannot form self-dimers, thus no odd numbered species.  Some of my gel results reflected the ideas I have just expressed, however some of them did not because the fiber A-B system was also forming distinct species in some cases meaning that the fiber motif is not as floppy as we imagined but possibly more rigid like the square motif.  Or else there is something suspicious going on with my experimental technique.

The start of a new experiment can be an exciting and nerve racking time.  When working with RNA molecules, there is a substantial amount of prep work that comes into play before an experiment can even be run.  You begin by studying certain shapes that RNA molecules can fold into.  Currently I am studying a right angle motif of RNA.  Based on the construct of study you can analyze the folding capabilities and assembly of the molecule by looking at the effects different point mutations have on the specific construct.  There are sometimes hundreds of point mutations that you could make, however you have to deduce which ones would cause the biggest change in the molecules assembly into a 3D structure.  Next you can design the different mutant forms of the wild-type right angle molecule of interest and design complementary DNA primers.  Then the DNA can be synthesized via polymerase chain reaction and the RNA synthesized by transcription of the DNA.  Hence, a good deal of work precedes the experimental testing of the designed RNA particles.  Which is generally why I am kept on the edge of my seat when I am awaiting the results of my first experiments.

I ran my first polyacrylamide gel electrophoresis experiments today in order to analyzed the assembly patterns of my newly synthesized RNA molecules.  I radioactively label the RNA molecules with radioactive phosphate in order to visualize where the RNA molecules migrate to on the gel.  After running the gel it is dried and exposed to a storage phosphor screen which can be scanned in order to give an image of where the RNA bands occurred on the gel.  We expose the gels to the storage screens over night, so I won’t know what the results of my experiment will be until Monday, which is even worse for my impatience since I will have to wait until the weekend is over to view my results.  The best thing about the entire process is that you can either get the results you were expecting and prove the concept you came up with originally or you could see something completely unlike what you expected which is equally or more exciting because you are discovering something new that did not go along with preconceived ideas.   Or maybe my experimental technique was a little off, but it will be fun to figure out the reasons behind whatever results are obtained.

Right as midterms were hitting I ran into other problems with my RNA synthesis.  It always tends to be the case that things spiral more out of control as the amount of things you have to deal with increases.  I was trying to increase my PCR (polymerase chain reaction) yield of the synthetic DNA I was amplifying in order to transcribe the complementary RNA molecules from it.  Although I had produced all 21 of my new RNA molecules via transcription I had some molecules with low yields, hence it would be difficult to carry out experiments since some of the molecules I will need to be in large concentrations.  So as the intense study sessions and cramming of biochemistry material into my brain began I also embarked on the RNA synthesis train which usually involves spending at least three or four days to complete the journey.  Once again it was a challenge to balance studying for midterms with working in the research lab and also taking a biochemistry lab course that consumes about ten or more hours of my time a week.  However I am beginning to master the tactics of planning my RNA synthesis steps around my class schedule so that I can either be in class  or studying while reactions are incubating or gels are running.  And it was definitely rewarding to figure everything out because I was able to re-synthesize my RNA molecules with some of the highest yields I have ever attained and in addition I was not brutally destroyed by any of my midterms; I think the first one went pretty well.