The gene I'm focusing on is called nitrate reductase. Nitrate reductase, or NR, is one of several genes used by diatoms to assimilate or take in nitrogen from the environment and make it usable by the cell. Nitrogen is often a limiting factor in growth of all organisms because it is so important in making DNA. Specifically, my project looks to determine where on along the gene it might be regulated by the cell.
|Transcription factors bind upstream of the open reading frame, allow transcription of the gene's DNA to begin. Transcription yields mRNA transcripts, which may be regulated at the downstream end by molecules in the cell.|
It's been shown in a few well documented cases that the end of the DNA that encodes a gene may be responsible for regulating the gene's expression. We're going to look at this specific region of the gene (in addition to the rest of it as well) to try to figure out if this is the case for this gene, which will open up a lot of other doors for this kind of research in diatoms (specifically with this gene).
Post-transcriptional regulation: modulating protein synthesis
In order to do this, we first have to isolate the gene of interest by using specialized primers. These primers will latch onto specific sequences and amplify a region of DNA exponentially in a process called a polymerase chain reaction. We then put this region of DNA into plasmids (my post on plasmids). The gene's activity can be tested in vitro ("in glass," like a test tube) or in vivo, within an organism (which is what we plan on doing in my project).
In my project instead of an entire coding region for a gene, we're using the untranslated regions of the NR gene. In lieu of the gene that codes for NR, we're using a reporter gene, or a gene that can be measured, which can be translated into gene activity. The pieces of DNA we're adding are the pieces of DNA that flank the coding region of NR. The reporter gene we're using is eGFP which glows green when expressed. Pretty cool, huh?
The plasmid I've been working on is a control plasmid for in vivo experiments. Instead of an NR terminator, we're using the terminator from a different gene (actin). We'll be able to compare the amount of eGFP being made by cells that have the NR terminator to cells that have the actin terminator. If this terminator region is an area where regulation occurs, we'll notice a difference in activity because the NR terminator will cause activity to decrease when appropriate but the actin one will not.
Building inducible expression plasmids
Diatom Genetic Transformation!
Diatom PCR Colony Screen
To summarize, the progress of my project so far has looked something like this:
- Develop inducible expression plasmids that express the reporter gene GFP, which will convey the activity of the nitrogen assimilatory genes nitrate and nitrite reductase.
- Genetically transform cultures of diatoms (spread on agar plates) through particle bombardment (plasmid DNA, coating tiny gold particles, fuse with the genomic DNA).
- Select for positive transformants:
- grow diatoms initially on agar plates
- select for cultures resistant to antibiotics
- transfer antibiotic-resistant cells to liquid culture
- screen antibiotic-resistant cell lines for presence of GFP through PCR