Sunday, February 13, 2011

Midi Preps & Stickleback Tanks

I had a fairly productive week, hence my lack of posts for 6 days!

In the lab I did a Midi prep of my Actin and NR plasmids. LOL WUT? -- Okay, so, a Midi prep is a higher scale preparation of plasmids from bacteria cells, which therefore yields more plasmid DNA than the Mini preps I've done in the past. While it's great that Midi preps yield more DNA for my experiments, they're definitely more tricky and take longer to set up.

Essentially, I have an array of plasmids at my disposal that will help us look at different genes associated with nitrogen assimilation. As I've said before, these plasmids have a promoter (Pnr), an open reading frame (ORF; gene), and a terminator (Tnr). For the most part, the ORF is the green fluorescent protein (GFP), which we use as a reporter (if the Pnr–and to some extent the Tnr–are working in the cell, GFP will be expressed and the cell will glow, which is easily seen!). The parts of the plasmids we have been changing around are the Pnr and Tnr regions of different nitrogen assimilation genes used in diatoms. The plasmid I made has the Pnr from NR (nitrate reductase) and the Tnr from Actin, which has no role in nitrogen assimilation. We can than compare the activity of GFP in vivo (in the cell) between diatoms that have the NR Tnr and Actin Tnr. If there are any differences, this would suggest that NR is regulated at the 3' UTR (at the end of the ORF) based on environmental conditions.

The control plasmid, with both an NR Pnr and Tnr.
So these Midi preps I did this week yielded me a significantly higher amount of plasmids, and I'll be using them to transform my diatoms. In order to do a Midi prep, I had to do a series of bacteria cultures that grow over night in out 37°C room. To start, I did what's called a glycerol stock streak. That is, I took 2 cultures stored in glycerol at -80°C and streaked them onto agar media plates. They grew overnight, and the next day I took a single colony from the plates that grew over night and mixed them into 5mL of liquid media in a test tube. Those guys then grew overnight, and on the third day I took a very small volume of them and poured it into 50mL liquid media in a beaker. Yeah, and then that beaker also grew overnight. In all, it takes three overnight cultures (glycerol stock on a plate, starter liquid culture, final liquid culture) to get what I needed for the Midi prep.
The Actin plasmid I constructed with the Actin Tnr

I took the 50mL of liquid bacteria culture (which is 16x the amount I used for a Mini prep) into the Midi prep, which after a first spin down of the cells, was a series of adding different solutions and filtering them through specialized syringes. The Midi prep uses cell and DNA chemistry to isolate the plasmid DNA through a series of steps. It's pretty neat! Because this was my first run (which I did with my professor), it was a little slow and took some getting used to. It definitely helps to have an extra set of hands, but I think I'll be able to do it okay on my own next time.

I did a grow up of bacteria cells containing both my NR and Actin plasmids and ran them through the Midi prep. From here, I'll most likely precipitate them and resuspend them into less liquid so they're at higher concentrations. These plasmids will then be used in my transformations. In addition to prepping these plasmids for transformation, I need to track down the plasmids with resistance to antibiotics. They're already prepped and stored in our freezer. These plasmids will be transformed along with the nitrogen plasmids, and will allow for us to select for diatom cells that have antibiotic resistance.

In other news, I got First Academic honors for last semester.
Niccccce! That's relatively exciting.

In other other news, we're starting our behavior experiment projects in Animal Behavior. On Thursday, we ran some very rudimentary experiments and set up our tanks for our Threespine Stickleback. We're going to be investigating foraging competition (competing for food) and hopefully get some cool results. Looks like we'll have to "starve" our fish long enough for them to be hungry. We want them to compete for food, not leisurely eat the tiny little worms we drop into the tank! It'll be interesting to see how our project progresses. Admittedly this project will add to stress during my week, and force me to get a lot more work done on the weekends. I no longer can have leisurely Saturdays! Good think getting a lot of work done makes me feel really good!

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