Resurrecting old lines of transformed diatoms

I've begun the process of resurrecting old lines of transformed diatoms for future use in experiments in my lab. These diatom cells are from the original agar plates used in the particle bombardment genetic transformations. Currently, I have about 30 discrete lines of diatoms, each with one of my four different experimental plasmid DNA constructs. I'd like to dramatically increase that number if the need arises. The goal is to have a giant pool of diatoms, separated by the type of DNA with which they were transformed.

The overall process is outlined below:

As I stated in the graphic, these lines of diatoms come from the original transformation plates I used to start the discrete lines of diatoms I have now. After using them, I placed them underneath the rack where I grow my diatoms. As this shelf is not a solid plane, limited light did reach these plates. However, the amount of light they did receive was very limited in comparison to the normal growth conditions. This was the only reason why these cells appeared to be alive when I looked at them again recently.

When I was selecting for lines of diatoms back in August/September, I first plated the cells from liquid culture (the original plates I'm discussing now) to be used in the transformation, scraped those cells into liquid culture (much like the diagram above), and then plated the cells again after a recovery period. These cells plated on fresh plates were then left in constant light after they were used to inoculate liquid cultures. After sitting in this light for months on end, they soon faded from their usual brown hue to white. These cells died.

But the original transformation plates, sitting in a dark, cool place, were still brown. Even though they had been sitting on plates without selection (and more importantly without the addition of fresh nutrients), they appeared to still have some life in them.

So I scraped off as many cells from each transformation plate and transferred them into liquid cultures, without any selection. At this point, I had four different test tubes, one for each of my different plasmid constructs transformed into the diatoms. After a week of surprisingly rampant growth, I decided to see if they were still resistant to antibiotics.

Which they were! YEAH SCIENCE!

This past week I have since transferred them to larger liquid cultures to allow the resistant clones to proliferate. I will then plate all of these cells onto multiple selective agar plates, and allow them to grow up before placing them in a cooler, darker place in the culture room.

Until I plate my cultures and select for single colonies (as shown in the last stage of the graphic), I will have "pools" of transformed diatoms for each of my constructs: two different constructs for the nitrate reductase and and nitrite reductase genes. Because of the random insertion of the plasmid DNA into the genomic DNA of the diatom from the particle bombardment, each clone we can separate from the rest of the pool will be distinct from all of the others. This means we have the potential of growing hundreds of different lines of diatoms given the opportunity (or from what was left on the transformation plates).

It will be interesting to see what I do with these lines. If I have time this summer and some money to support me, I may try playing around with different culturing techniques to bolster a manuscript to submit to a journal.

Sometimes science isn't linear (and I'm not talking about exponential growth)

Sometimes science (my project) isn't linear, and I've been getting caught up in this recently with my posting.

I wanted to do a series of posts and videos on the process of transformation and the completion of my first NiR plasmid, but what I found was that sometimes things don't work out the way you want.

So, let me backtrack a bit.

As I mentioned before, it appears I have transformed bacteria colonies that have my PCR insert. Great! But I've been having trouble getting the insert to amplify out of the plasmid once again. I grew up several bacteria colonies that looked like they had my insert (white colonies on X-gal) and performed a plasmid prep that yielded very little plasmid DNA. I need a decent amount of this plasmid to allow me to digest (cut) out and obtain the insert.

Now, I'm trying to do yet another PCR reaction in a much larger volume (50µl rather than 10-20µl) using the plasmids I obtained from my lame plasmid prep. If this works, I'll have a lot of copies of the NiR terminator insert, which I can then slice off the ends with restriction enzymes. Then, the insert would be ready for the next step.

That is, if I can get this to work. :-\

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!

More Plasmid Work

I've already talked about how I've made an inducible expression plasmid to test the mRNA stability of nitrate reductase (NR) transcripts in vivo in the diatom Thalassiosira pseudonana. The plasmids run by using a promoter and terminator sequence to run the expression of a reporter gene (we're using GFP). We have a set of NR plasmids, one with a terminal region of NR and one with a terminal region of action (which has nothing to do with nitrogen assimilation). The former plasmid should mimic endogenous activity, whereas the latter plasmid should not mimic the normal conditions in the cell. NR reduces nitrate (NO3-) to nitrite (NO2-), which is then reduced to ammonium (NH4+) by nitrite reductase (NiR). As such, in the scheme of nitrogen assimilation in diatoms, it makes sense to test the regulation of NiR as well.

To do this, I've started work on a set of NiR plasmids. They will also run the inducible expression of GFP, but with their own promoter and terminator regions. Both will have their NiR promoter region, whereas one will have the NiR terminator and one will have the actin terminator region. Both sets of plasmids will be transformed through particle bombardment and then in vivo expression can be measured (through GFP activity).

As it stands, we have a plasmid that contains the NiR promoter & GFP but not the proper terminator. The NiR terminator was cloned into a separate plasmid as part of the process in amplifying out the terminator region of interest. Currently, I'm trying to amplify the terminator out of the plasmid, and in doing so add restriction sites to the plasmid.

The cartoon below represents the series of steps that we have to do in order to manufacture the desired fragment of DNA with restriction sites at the beginning and end of it, which allow us to easily cut out the DNA fragment and place it into a plasmid. Right now, the NiR terminator is represented by the red box. It was amplified out of the entire NiR gene to yield a small piece (between 500bp and 1kb) of the the gene. This piece was at the very end of the open reading frame and extended past the 3' UTR (the terminal region). This was done in a PCR reaction, and the PCR product was inserted into a vector, transformed into bacteria, grown, and then the plasmids were isolated once again the yield the below plasmid.

What I want to do is amplify a smaller portion of the insert out of the plasmid, and make a bunch of copies of it through a PCR reaction. A second set of primers (the green lines) will amplify within the region of the insert, while adding restriction sites. Currently, I've having problems getting this PCR to work because less than half of the primer fits to the DNA of the current insert, while the other half is going to add the restriction site. However, I changed the protocol for the PCR reaction I'm running at the moment. I changed the annealing temperature for the first 10 rounds of my PCR and then I'll bring it back up to what I ran it yesterday (a PCR reaction that did not work, lanes 3-5; right--below right my PCR samples loaded; the faint blue samples are the ladders I used, lanes 1, 2 & 8; the red samples are my PCR reactions that used Coral Load, a special PCR buffer/loading dye combination, lanes 3-7).

...NiR terminator amplification to be continued...

Back to Science

While most of my semester was dominated by finishing up my undergraduate courses, I did get a little bit of work done on my "Monitoring in vivo transcription in the marine diatom Thalassiosira pseudonana using eGFP reporter plasmids" project. Most of my work can be summed up through my post on working on the culture side of the transformational protocol (another post here), setting up media for the transformation protocol, and working on my Academic Spree Day poster and a proposal for my summer work on my 5th year Master's project.

I've had really good success on multiple science projects by putting a lot of effort working on a poster or Powerpoint presentation and then writing my term paper for the project. Below is the final draft of my poster (which I hope to hang up outside of the lab later today), which I presented at Academic Spree Day (ASD).

I had a lot of fun at this year's ASD presenting and sharing my work with science (and non-science) professors and classmates. It was particularly satisfying sharing my poster with fellow science (but non-biology) students, and then going over their posters with them. Certainly a little bit of sharing and learning!

This poster then was the basis for updating my project proposal:

This week is my first week after graduating on Sunday. The name of the game for this week is to get back into the flow of things in lab, and hopefully establish a workable rhythm. At the moment, I'm continuing to work on creating inducible expression plasmids for nitrite reductase.

Spring Break -> no classes -> I can update once again

Classes have been killing me.

I haven't been this busy in a long time. It seems like every week is finals week! I haven't been able to catch a break until this week, which is Spring Break. *catches breath* I don't even know where to start because I haven't done a YouTube video in a month or a real post here in weeks.

Well okay, so this week is spring break which is really nice. How am I spending my week off? Well, my friend Jesse from Pennsylvania is coming up to visit for a few days. I'm wicked stoked he's visiting. He's one of my best friends, and I met him in Australia. Jesse and I were in the same study abroad program in Perth, which I've chronicled in my study abroad blog. Besides his visit, it's catch up time for me. Hopefully I can get a head start on the remainder of the semester, which will be nonstop until May 10th or so. Oh boy. Can you tell I'm excited!?

This will be a long post for sure, so please bear with me.

Directed study

Over the past few weeks, I've been doing culture practice. Because we will be transforming diatoms on plates (to the right), we need to be determine a protocol for growing diatoms on the plates and transferring them to "native" liquid culture. (The top picture are the plates with a poorly drawn circle within which I plated the diatoms [below]. I had to centrifuge down 40mL of culture for each plate, something like a hundred million cells per plate.) I need a little more practice plating the diatoms in the circle outline, and this will ensure maximum efficiency once we do the actual transformation.


In order to plate the cells, I have to count them like I mentioned before. I can then plate a known estimate of cells, and determine what works best. Once I plated the cells and determined how long they took to grow and how few cells I could plate in order to see cultures grow, I needed to transfer them back into liquid culture. This is just like how I grow bacteria on plates and transfer them into liquid culture. But, because the liquid culture for diatoms are much larger than bacteria cultures I use, we need to start the diatoms off in a very small amount, like a few mL. To do this, I took a wire loop and removed a single colony (several hundred cells) and placed it in a 1.5-3.0mL seawater well, on a 6 well plate, which you can see on the middle right in the picture below.

After a few days when I got visible growth, I transferred them into a 5 or 10mL culture, seen in the test tubes. By making larger and larger cultures, we can make sure cells are growing well. If we put our initial cells in a half liter flask, it would take up to a week or two to discern whether we got growth or not. But by growing them in small volumes, we can make sure we're doing okay sooner. (In this picture here you can see my four different diatom culture stages: the plated colonies, bottom left; the test tube 5 and 10mL colonies, top left; the 6 well plate containing 1.5 and 3mL colonies, top right; and the trial transformation plates on the bottom right.)

After break, my professor and I hope to travel down to Rhode Island and transform my diatoms. I can't wait to finally move forward with this project! I'm hoping my post-transformation project will really speed up and I can start collecting data and maybe publish something!

Last but not least,  I was finally accepted into the 5th year biology program, which is really exciting! while I had little doubt I would be accepted, getting the official letter was pretty cool and relaxed me a bit. I got my letter last week, months after other 5th year programs decided whether students could continue their projects or not.

Animal Behavior

A side from reading what I consider to be a lot of papers on different aspects of animal behavior, we're slowly starting to begin our research projects. We have half a semester to collect as much data as possible, write a sophisticated lab report/research paper, and create a lengthy presentation and poster. Something tells me it's going to be an incredible crunch, which is why I'm so eager to get as much studying done as possible this week.

Something I drew on a whiteboard during class...

My research project along with a few friends is to investigate foraging competition among threespine stickleback juveniles, which we call fry. The biggest lab on campus uses threespine stickleback for an array of studies, mostly concerned with evolution and adaptive radiation. But yeah, our project. We're looking into whether body size affects how well stickleback fry can compete for food. In ponds and lakes with limited food sources, competition is likely to be high and we're curious if size is an advantage.

To look at this, we'll be feeding pairs stickleback fry limited amounts of bloodworms, and record their competitions. We do this by pipetting bloodworms into a small tank, and videotaping the fish activity. We can then go back and watch their interactions and analyze it.

 

However, in order to get them to be competitive, we have to make sure they're hungry... so we don't feed them for half a day before testing.