Week 4

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. 

The fnr protein is a transcriptional activator that binds to certain sites in the narG promoter, helping RNA polymerase to bind and intiate transcription.  In B. subtilis, the narG gene has one fnr binding site in its promoter region, while anthracis has two (or so we think).  Anthracis also expresses the nitrate reductase gene under aerobic and anaerobic conditions, despite the fact that using nitrate as a terminal electron acceptor when oxygen is present is energetically unfavorable.  Also, in subtilis the fnr protein is stabilized by a [4Fe-4S] complex, which allows it to bind to the narG promoter.  Under aerobic conditions the iron-sulfur complex degrades, preventing fnr from binding.

With that background information out of the way, I'm currently working on testing whether or not the second putative fnr binding site is responsible for aerobic transcription, which I'll probably talk more about next week when I've gotten a bit further in the work.