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Week 7

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.

The lac operon, a set of genes for lactose utilization, is one of the classic examples of bacterial genetics. In addition, the activity of the lacZ gene, which encodes the enzyme beta-galactosidase, is fairly easy to monitor (all it requires is a fairly simple reaction). So, the lac assay is a highly useful tool to determine the relative level of transcriptional activity of a gene. The promoter of interest can be fused to the lacZ gene in a plasmid vector, and the level of activity indicates the level of transcription of the gene in question.

Going back to my project, we used lac assays (under aerobic and anaerobic conditions) to monitor the activity of our assorted narG promoters. If everything went as we expected, only the first strain (carrying the intact narG promoter) should show aerobic activity.  Unfortunately, we found aerobic activity in one of the strains carrying a deleted FNR-1 binding site. We tested two more strains and found similar results, and used PCR to confirm that the correct fragment was there (it was). So, we now need to have the insert sequenced to see what happened.

In other news, my mutagenesis of the narG promoter was successful. We can now test our second hypothesis, that nucleotide spacing is responsible for FNR binding and aerobic transcription in the narG promoter.