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.

Staving off a massive diatom culling

At the end of last semester, chaos tore through my portion of the laboratory when a massive die-off of diatom cultures was a major possibility.

My diatom cultures have been growing in glass test tubes with liquid media, within which they're happy for an upwards of three weeks before they need to be transferred to fresh media. There are a number of factors which make culturing my individual lines of diatoms different from our various stock cultures of diatoms that also grow in the culture room, so it's been a bit of a change for me.

With a few weeks left in the semester, I inoculated two large cultures for my final experimental assay. After about five days, there seemed to be almost no growth, but I figured I'd give them a few more days. After a week I told myself I must have messed up somehow, so I inoculated two more large cultures and I was on my way. At about this time, I made some solid media plates and spread all 40+ of my cultures on individual plates to preserve them while I was away on holiday. (The solid media plates combine the sea water/nutritional supplements I usually grow the diatoms in with agar to solidify the mixture. This is essentially the diatom version of the plates I used to grow my bacteria on when I was cloning DNA.)

But then the second round of large cultures didn't growth either. (Meanwhile my plates still had another 5+ days to show any signs of growth.)

...Yeah... It was at this point I went into a deep panic because my diatoms in my test tubes were dying off and it came to my attention that the materials I was using to supplement my media (i.e. the nutrients to sustain growth) were, well, bad. (This is what happens when I'm not responsible for all of my materials. I am not attacking anyone, but when things matter in the lab I should really do everything myself like make the materials needed for my cultures.) This meant the plates I had made using these materials would not sustain any growth and all of those plates would have to be thrown away.

Now I was upset for multiple reasons: I lost a full day's worth of tedious lab work setting up my first 40 plates and all of my diatom lines were thinking about kicking the bucket.

GREAT.

Additionally, I didn't get in my final experimental assay because my diatoms kept dying in the large cultures due to poor nutritional content.


The end of the semester resulted in frantically making new plates with different materials and pleading with the science gods that they grew while I was away at home.

(Insert a lot of World of Warcraft playing at home)

After a week in the hills of Vermont and New Hampshire, I checked back into lab before heading off to Florida. Thankfully, almost all of my plates showed signs of life which meant I could catch my plane without fretting about my meager diatom cultures.

Attempting to save all of my discrete diatom lines.

After I got back from Florida a week later (roughly two weeks after the second plating), it was time to check on my plates once again.

This time I wasn't as happy with what I saw. Because I was hasty in finishing my plating before I left for home, I did sort of a good-enough-but-crude job. While some of my plates may still be good and support/sustain healthy cultures after they dry off some extra moisture from the initial plating (I sealed each plate to prevent them from drying out), I decided to transfer each culture back to new test tube cultures. I ended up using a combination of plated cultures (two weeks old), old liquid cultures (one month old), and really old liquid cultures (two months old), to sustain almost every line I started with. There are still a few lines I may be able to salvage from my plates
 

A majority of my massive diatom culture collection.

This has been an interesting experience for me and I still have a few things to learn and figure out. The tricky part is that I don't have a lot of spare lab time to tinker around and perfect everything. But because I like everything to be done right, I'll figure them out. First and foremost, I'd like to plate my diatoms so they can subsist for at least three months without me tending to them. While it will be a fair amount of work invested at the beginning of the process, it will save me heaps of time in the long run because I won't have to cater to the high-maintenance demands of my diatoms.

Once I settle back into my hectic life. I hope to consolidate all of my diatom cultures for long-time storage and press onwards to finishing my project for once and for all.

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.

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.