Last month my
undergraduate/now graduate research project at
Clark University reached a new level of awesome with the
transformation of diatom cells with plasmids I've been working on for several semesters. It's been a long time coming for sure, with both problems encountered in the lab project and juggling other college courses.
In my blog I've chronicled some steps along the way on my project, and I've talked about:
And now I can talk about the next step of my project: the transformation of diatoms with the plasmids I've created.
Now I am undergoing the process of
selecting lines of transformed diatoms for the next step in our project. But we've recently realized that the light intensity in our growth chamber is much brighter than previously published experiments growing transformed cultures of diatoms. Increasing light intensity reduces the amount of chlorophyll made by plants and algae. Why is this important? Two reasons:
- Less chlorophyll will reduce the pigmentation of our cells, making them harder to see once we transfer them to liquid culture.
- Our gene giving resistance to our antibiotic (that allows us to select for positive transformants) is driven by a chlorophyll-associated protein, which means expression may be reduced in higher light intensities. If chlorophyll expression is reduced by high light levels, then this may reduce the activity of the chlorophyll-associated protein that drives the antibiotic resistance in our diatoms, which means less resistance to the antibiotic in the media. This ultimately means reduced or no growth.
Now, our diatom cultures have been growing okay on their agar plates, but I'm beginning to wonder if they're growing slower in liquid cultures, where they're more likely to absorb more light. To combat this, I'm growing some liquid cultures behind layers of porch screen to reduce light levels penetrating the liquid cultures. In the below picture, you can see the different diatom cultures I have growing right now:
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| Transformed diatoms grow on agar plates (left foreground) an in liquid media (left background and right). |
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I've been growing diatoms in 3 mL cultures in little dishes, which you can see in the back left. Groups of 3 dishes sit in a petri dish to allow easier transportation. These dishes however require 3 mL of culture just to cover the entire bottom of the dish, which means the cells are going to be fairly diluted. I also started 1 mL cultures in the test tubes on the right, where porch screen blocks out a lot of the incoming light. Some of the 3 mL cultures also have screen on top of them to block out some of the light.
Aside from trying to grow our cells in liquid culture, I've been looking at them under a fluorescent microscope to try to determine whether our cells are fluorescing due to GFP expression or whether it's residual glow from chlorophyll. UV light from the microscope excites all pigments in the sample, and then I look at the sample through different filters that only allow certain wavelengths of light to show through. However, our current filter set up hasn't allowed a clear delineation between GFP and chlorophyll yet. Most of our pictures look something like this:
...but they usually have a lot more background color in there too.
My next steps will be to either design or order commercial GFP GSPs--gene specific primers for GFP. That way we can run a PCR on some diatom DNA and determine whether they have the GFP gene they are supposed to be transformed with. Because I have a great control (the original plasmid DNA), I can test a whole bunch of diatom colonies at once and be able to select appropriate lines of diatoms faster.
