Bill Odette's blog

I presented my work today-overall it went pretty well.  Unfortunately, I didn't draw many definite conclusions from the work I've done this summer.  It appears nucleotide spacing is not responsible for aerobic narG transcription, and the role of the putative upstream Fnr-binding site is still unknown.  However, the summer as a whole was great-I gained a ton of practical lab experience that I'll be able to take with me to school and beyond.

Wrapping up. One of our Bacillus strains carrying the Fnr-1-deletion narG promoter showed no aerobic lacZ activity (what we expected), while the other strain did show aerobic activity.  Two more strains we tested showed the same results.  Unfortunately I won't have time to do everything required to determine why this happened-I'll be performing another lac assay monday and tuesday to test the hypothesis that nucleotide spacing is responsible for anaerobic narG transcription, finishing my presentation, and that's about it.

Not a whole lot going on now, I've finished all of my plasmid constructs and have transformed them all into B. subtilis. I'll be doing one final lac assay Monday-Tuesday for the narG promoter containing a nucleotide insertion-I won't have time to finish everything and I really want to see if this one turns out to be correct.

My last few blog entries probably seem a bit cryptic, but it's been hard to figure out what exactly to put down. Going back a few steps-one of our original hypotheses was that the FNR-1 binding site of the anthracis narG promoter was responsible for aerobic nitrate reductase activity in anthrax.  We isolated six strains of B. subtilis carrying the anthracis narG promoter (two with both binding sites intact, two with one site deleted, two with both sites deleted).To determine whether or not this promoter was active, we used a lac assay.

Not much to report this week.  One of my Bacillus strains gave some odd results in our second lac assay, so I've been working on re-transforming and screening a new strain of bacillus.  I've also been working on transforming a mutated narG promoter into E. coli, without much success (although hopefully today will change that).  My new Bacillus should be ready by tomorrow, which means I can repeat the lac assay and see what's going on.

Week 5 was pretty busy.  We did two lac assays-the first seemed to support our hypothesis, but there were some strange results on the second, so we're taking a few steps back this week and looking at our Bacillus strains.  I also used two-step PCR to introduce a mutation into the anthracis narG promoter.  If everything goes as planned, we should be able to sequence the (hopefully) mutated promoter and continue from there.

This week has been much more productive than the last few.  I've added a picture to help illustrate what I did to the plasmid that was giving me so much trouble (pDH32).  After that, I was able to successfully ligate all three of my fragments of interest into another plasmid, pDG793.  The three fragments I've been working with are all segments of the B. anthracis narG (narG is part of the narGHIJ operon, which encodes a nitrate reductase) promoter region. 

On our first day here, an article was handed out on the importance of "stupidity" in science, realizing that the purpose of conducting research is to answer as-yet unanswered questions, and in order to do that we need to be comfortable with our "stupidity."  After the fourth or fifth time I failed to isolate and purify several DNA fragments (a process that takes, on average, several hours) it was all I could do to hope that I was the right kind of stupid.  However, thanks to the wonderful people at QIAGEN, I was finally able to isolate my DNA using a PCR purifi

This week has been frustrating. My project revolves around a stretch of DNA, and determining how exactly that stretch of DNA regulates gene expression. Before we can determine that, however, we need to isolate and clone several fragments containing different lengths of the DNA in question (the B. anthracis fnr promoter region). I've been trying to isolate these fragments this week, but have run into several problems with the plasmid vectors.

 Week one is almost over, and it's been interesting to say the least.  I've learned a lot of practical techniques this week including colony PCR, plasmid DNA purification, gel preparation, etc.  I'm still trying to wrap my head around exactly what my project will entail, but it's becoming clearer every day, and I'm excited to get into the meat of the work.  I'm running my PCR products from yesterday on a gel as I type this, so hopefully there will be some positive results.