Building inducible expression plasmids (aka Science)

Below is a video I've been trying to publish for a long time coming this summer, but because my summer project only came to a close this week, I've been hesitant to post anything.



In this video, I give an update on my lab work and discuss my overall lab project, which is a great review for myself and my viewers. This is an exciting time for me at Clark University, because I’m beginning the real stages of becoming a graduate student in our accelerated Master’s program. After conducting undergraduate research, I’m using it to support a 5th year of schooling at Clark to finish up my Master’s.

My project concerns the 3’ UTR of the nitrogen-assimilating genes nitrate reductase and nitrite reductase in diatoms (if you haven't read up on my blog before), because we have evidence that along this region these genes are up- and down-regulated by environmental conditions. To test this hypothesis, I’ve created plasmids that drive expression of a reporter gene (GFP) with promoter and terminator regions derived from diatom genes. We’ll compare the activity of GFP with differing 3’ UTR elements to determine whether this region of the nitrogen-assimilating genes is crucial in their regulation.

For the gene nitrate reductase, this is old news. But it is news for my blog that I've completed the plasmids for nitrite reductase, which is really the story of my video.

I'm really excited to be where I am in my project. This week, I hope to genetically transform my diatoms with the plasmids I've created. Oh baby Darwin, this could be a HUGE week for me and my scientific career! I'll be back soon with transformation news.

What's up graduate student office?

I grew up liquid bacteria cultures last night so I could harvest their plasmids this morning, but alas, my liquid cultures didn't grow overnight. They looked this morning pretty much like they did last night:

I grew four different cultures last night from the same bacteria colony. This colony I has semi-recently used for a plasmid harvest (plasmid prep), so I was shocked to see they hadn't grown. On the left (the darker cultures) is 2xYT buffer (2 times the amount of Yeast Extract Tryptone), an especially nutrient-rich media, and on the right (the lighter cultures) is the standard LB broth (which I just learned from Wikipedia is commonly incorrectly called Luria broth). I set up one of each culture at 1 volume and 2 volumes of the antibiotic ampicillin. I usually grow my cultures at 2 volumes (100µg of amp per milliliter). The amount of antibiotic is very important--let me explain: we're growing these bacteria for their plasmids (sort of like growing people for organs, like in the movie The Island--sorry if I spoiled that one for you hahahaha), and the plasmids have a gene for antibiotic resistance in addition to the other parts of DNA we're cloning the plasmids for (click here for a 101 post on plasmids). Therefore, any bacteria that have a plasmid should be able to survive in the presence of ampicillin. This means we need to add ampicillin to our cultures, to weed out the bacteria that don't have the plasmid. However, if we add too little ampicillin, some bacteria that don't have the resistance gene (from the plasmid) may still be able to survive. If we add too much ampicillin, none of the cells may survive. It's a catch-22: too many bacteria cells (especially those without the plasmid) due to no or too little ampicillin, will give us poor plasmid harvest yields. (This is particularly problematic because bacteria without the plasmid may end up growing faster than counterparts with plasmids, because they don't have to invest the energy into making the plasmids.) However on the other end, too much ampicillin will prevent the growth of cells and I won't get any cells to harvest their plasmids.

In my case, my bacteria colonies were probably too old to start new liquid cultures from. Bacteria colonies are best used if they are actively growing, because they are their healthiest at that point. We'll have to see if I can get these current colonies to grow anymore.

In other news, I'm using my new graduate student desk (which I'm sharing with my labmate Jessica):

It's pretty cool to have a space in the graduate student office, because now I finally feel like a graduate student at Clark. Just settling in as you can see. Jess and I will be using this space primarily to do our reading and writing, but it could also be used to plan out experiments, do research, etc.

What's up PCR reaction that finally worked!?

BAM! LOOK AT THOSE BANDS!

Three PCR reactions, three beautiful bands. Looks like digesting the insert and then amplifying it really did the trick. This means I can use my PCR reaction to transform some bacteria!

The idea is to insert our PCR reaction into a plasmid vector, which we then transform into E. coli. We do this via heat shock, which causes the bacteria cells to take up plasmids. We then will grow the E. coli, conduct a plasmid prep to collect all of the plasmids they grew, and then I will perform another digest to recut out the terminator region (the PCR product). This way, I can be sure the restriction sites were correctly added onto the terminator region, which I need in order to insert it into the rest of the plasmid I already have.

I'm transforming the cells as I write this, and will be back soon to give updates. Yay science!

Oh Snap! New Plasmids!

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!