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Sink or Swim [week#1]

This has been a very difficult week for sure.  Much of the week has been spent figuring out what exactly we are investigating. A lot of the time between my experiments, meetings and the epic two hour bus rides to and from CMOP has been spent reading background papers and Sushma's Thesis (much of our works centers around completing experiments she has left unfinished).  Thankfully, I finally think kind of I know what I am doing for the next nine weeks, and honestly, it kind of scares me the amount which I am trying to accomplish in such a short time.  I am excited to give it the old college try though :)  Also, I am working with a high-school teacher and student on this project, and have enjoyed explaining a lot of the theory behind WHY we are doing what we are doing, and WHAT it means.

So far, I have been trying to generate expression plasmids (with PDG793) which couple the SdpA and YkuN promoter regions to the LacZ gene. A lot of the processes involved I have done something akin to them in the labs at PSU, but the specifics are very different and more complicated (aka AWESOME to learn).  There have definitely been some bumps along the way.  Various errors from one of the three of us have slowed our progress a little (aka needing to redo the first PCR a second time due to pipetman errors, etc), but we have thankfully created a working plasmids and on-time at that!

Our research resolves around trying to figure out the class II, Nitric oxide sensitive, transcriptional control of the SdpA[BC] operon (SpdA is the sporation delaying protein) and the YkuP[ON] operon (YkuN is the Flavodoxin protein).  We are doing this by trying find possible direct and indirect interactions with the promoters of these operons have with the ResD (a two-component signal protein with ResE) and NsrR (the Nitric oxide sensor) transcription control proteins.  We are conducting this study in an in vivo instead of an in vitro environment because the transcription control at these class II binding regions are probably topographic and require the plasmids to be super-coiled for proper binding and regulation.

This research relates to the CMOP/OGI over-arching goals because it explores a fundamental understanding of these processes on a cellular level, and works toward the goal of developing a holistic understanding of Bacillus subtilus.  Also, it may contribute to strengthening our general understanding of multi-component transcription controls, how DNA-binding transcription proteins possibly have a duel function of changing the morphology of DNA in prokaryotes (like histones in eukaryotes), and finding novel DNA binding motifs for class II promotion.

I didn't intend to mislead above, but I wrote this blog about 3 hours before the end of my day and it didn't quite end as I had hoped...  I had an unfortunate and odd error after the second restriction endonuclease digestion of my plasmid fragment, and thus I wasn't actually able to complete my expression plasmid before the weekend.  I worked extra hard on Monday though and was able to redo the work necessary to get this done.  So, I am a day behind where I would have optimally been had I not had this error.  I was extremely frustrating last Friday with my sour and difficult to explain result (somehow the plasmid either was cut at a different site or recircularized), but getting a positive gel at the end of Monday was very relieving for sure.  Hopefully, I don't have any more wasted days due to similarly mysterious errors.