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One down & three more to go (week 2)

Yay!  This week went fairly smoothly and without any major surprises (well, for the most part...).  I spent Monday redoing about three days worth of work from week one, all in one day.  Very frustrating that I had to do it, but great practice and I thankfully was successful this time at creating the expression plasmid.  The rest of the week in the lab centered around creating a competent E. coli colony and transfecting into it my first expression plasmid for replication.  This also involved growing the E. coli in ampicillin enriched media to ensure that only cells which had my plasmid were able to survive, and running a colony PCR to ensure that it was my plasmid which was providing this resistance and that the promoter insert was the correct length.  We ran into a snag when we attempted to run the colony PCR with Dynazyme polymerase enzyme, however, but were able correct this by switching to the more expensive Gotaq enzyme.  Once the colony PCRs verified which clones contained the plasmids we desired, we chose three and placed them into 2xYT growth media for the night.  Finally, today we were able to isolate the now cloned expression plasmids from the E. coli host, and send a sample of one of them to be sequenced at the OHSU primate center.  The sequencing is important because it ensures that we are not working with expression plasmids which were mutated during their generation (aka via polymerase errors, mutagenic cross contamination, etc).

While all this was going on, we also attempted to begin generating some of the shorter promoter sequences (w/5' deletions), which we will require later to determine the regions of the promoter region which are important for their interactions with the various proteins involved.  I worked on generating the two shortest segments of the SdpA promoter sequence, while the high school intern, Felicia, worked on the longest deletion segment.  Unfortunately, I ran into a snag with the initial PCR with one of the deletion segments, in that the primer probably didn't work properly.  So, I was only able to get one to the expression plasmid generation stage by Friday.  Felicia was also successful with her segment.  Keifer, the high school teacher intern, was also successful at making a deletion segment for the YkuN promoter sequence he is working with.  Hopefully, we will be able to get these finished and ready for sequencing by next Friday.  Also, we are hopeful that we will be able to get the unsuccessful deletion primer to work on the first expression plasmid with a better success rate than we had with chromosome DNA.  However, we will have to wait to do this till after the sequence data is generated to ensure that plasmid is correct.

Last Thursday, I had the pleasure of viewing the NSF site visit.  It was interesting to see all the work being done by the center, and to get a chance to talk with one of the NSF representatives about how the whole process works for gaining financial support for research.  Very interesting indeed!  I wish I could have been able to witness all of the presentations and gain a better understanding of all CMOP is doing, but I had to get some procedures done in the lab that day to ensure I could send my plasmids to sequencing by this Friday.  I'll just have to make up for it by spending more time reading the descriptions on the website.  My interest was definitely piqued though!  

So there you have it.  I think that pretty much covers all that went on this week.  It has been a lot of work for sure, but I have definitely enjoyed the challenge and do feel that I am getting into a better rhythm with things.  I am excited to get to the real testing when I have finally generated all the Bacillus subtilus mutants, but I will have to wait a few weeks before that happens.