This summer's project has been to complete inducible expression plasmids to test the regulation of nitrite reductase (NiR) in the diatom Thalassiosira pseudonana. Inducible expression means we can induce the expression of a gene located on a plasmid based on environmental factors. These plasmids will be used to test whether the 3' end of the NiR plays a role in regulation the gene's activity based on environmental cues. We'll compare the activity of our reporter gene (GFP) from our control plasmid (with an NiR terminating region) and our developing experimental plasmid (with an Actin terminating region, as actin has no role with nitrogen assimilation).
An analytical double digest and gel to determine which of my bacteria colonies actually had the plasmid I wanted. My colony screen gave me some funny results, so I wanted to double check with a digest. The digest confirmed several of my colonies appear to have the plasmid and correct insert, giving me the green light to grow up more bacteria cultures and prep and purify their plasmids.
While it appears my NiR-NiR plasmid is completed (sections of the NiR gene flank the reporter gene in the plasmid), the completion of the experimental plasmid is right behind the control plasmid. Today I transformed some bacteria with what I hope is the complete experimental plasmid. I should know by tomorrow afternoon if I have what I think I do!
Meanwhile, I have scheduled a date at the University of Rhode Island to transform my diatoms! While I may only transform the diatoms with my set of nitrate reductase (NR, a different but related gene) plasmids (both with an NR terminus and an actin terminus, like the NiR plasmids I'm finishing up now).
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