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.

Counting cells and taking names

I spent a good portion of Friday morning and early afternoon in lab doing some work and figuring out where to go next in my project. I talked about different kinds of reporter plasmids we may use in this project with my professor (we may incorporate other genes that have plasmids already made) in addition to what kind of antibiotics we're going to use.

Two types of antibiotics have been used in recent projects transforming diatoms: nourseothricin and zeocin. The former is 100 times less toxic than the former, so we're going to start with using that.  Right now we're growing the plasmids without any selection pressures (no antibiotics). After we get a feel for how well the cells grow on the agar plates, we'll test them with nourseothricin. First of all, we need to make sure nourseothricin kills our diatom cells. This way once we transform them with our plasmids (which will include the anti-nourseothricin plasmid, nat), we'll know that only diatoms that have been transformed with nat will survive when exposed to nourseothricin.

In order to grow the diatom culture on an agar plate, I had to count them first. This entails pipetting a small amount of diatom liquid culture onto a hemocytometer, which is essentially a microscope slide with gridlines on it. The hemocytometer allows us to estimate the number of cells per milliliter. That way we can know about how many diatom cells we're putting into a fresh culture or agar plate. I'll have to get a hold of a special camera or something so I can take pictures of what I see on the microscope!

Once I counted my cells, I plated four different amounts on four different plates. This way, we can see how different starting amounts of cells affects how they grow. These cultures will take up to a week or so to grow, so I'll have to find something else to keep myself occupied in the lab in the meantime! :-p

After they've grown up a bit, I'll start testing the nourseothricin on the cells like I mentioned above. We could do this by soaking paper discs (about the size of hole punches) with antibiotics, or spreading antibiotics on a plate and then growing them.


I talk about all of this stuff in my video blog post below:



Outside of the lab, in the so called real world, we've been having a slight thaw. This has been warmly welcomed on campus. It rained most of the day yesterday and today it is in the mid 30's. I was wondering to myself the other day what would happen if temperatures jumped up into the 60's for two or three days. All of this snow... would lead to massive flooding. How awesome would that be!?