Time has flown by at OIMB! I can't believe our last week is coming to a close. It's been an amazing experience and I can't believe it's almost over. This week in lab has been a whirlwind; I've been trying to test out my 'Cat' and 'Dog' probes with varied degrees of success. My 'Dog' construct seems to be fairly unstable and expresses inconsistently in both starfish and barnacles with little localization. My 'Cat' construct, on the other hand, appeared to cause high levels of Rho activation, especially during second meiosis. However, since I injected the 'Cat' probe along with a cocktail of other probes, I couldn't see the 'Cat' expression alone. Instead, I had to judge whether other probes had a reaction to my probe (almost as if my probe was a treatment, not a fluorescent tag). Ultimately, more experiments are needed to better understand whether the probe is activating myosin and promoting contractility. Although I won't be here to help, hopefully my probes produce useful results in future experiments. Hiking some of the South Slough trails with Renee and Megan! We are having our final poster session this week as well. Although it was definitely a challenge to integrate everything I worked on this summer into one cohesive final product, I'm fairly happy with how it turned out. Despite my ‘Cat’ and ‘Dog’ probes not quite working as planned, my poster is a reminder that I did have some success with other probes this summer and did get some interesting results. I'm going to miss juggling oocytes between microscopes, babysitting eggs during long recording sessions on the confocal, poking at nudibranchs in the sea tables, bonfires at the OIMB beach, and most of all, the people I've met. We've formed a tight-knit community here at OIMB, and it's definitely going to be hard to leave. Thank you to everyone who helped make this summer so special!
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After testing out some of my probes last week and determining that they are lethal at high concentrations, I took a different approach to injecting them in hopes of more informative data. First, I injected the probes at lower concentrations. This experiment produced more informative results; I found that although the oocytes still don’t go through anything resembling meiosis, they don’t explode (as often). Additionally, as expected, oocytes experienced high Rho activation throughout, almost as though I had unplugged something important that was inactivating Rho before… This period of high Rho activation made the cells turn completely white, since the Rho probe lit up all over the cortex. This led to the mutant's current nickname: 'whiteout'. As mentioned above, unfortunately, not all oocytes remained unharmed during the duration of my experiments. After addition of hormone to those oocytes injected with high concentrations of either my PBR or Snowflake-PBR probes, I found that the oocytes would try to contract as if they were trying to start meiosis, but would fail quite dramatically. A video is attached – warning, it’s a little gory! (sorry for the low quality)
Apart from accidentally exploding some oocytes this week, I have made good progress on other aspects of my project. My ‘dog’ (Rock regulatory domain) is almost ready for injection, with the ‘cat’ (Rock kinase domain) not far behind. Also, the pieces (front, middle, and tail) of the S. purpuratus Rock are ready for Gibson assembly: a cloning method that allows for assembly of multiple small fragments into a single cohesive sequence. This will allow us to finally construct a probe specific to an echinoderm Rock. Although I probably will not have a chance to include ‘cat’, ‘dog’, and the S. purpuratus data on my poster I will still try out these probes before I leave in hopes of confirming my preliminary results. Outside of lab, OIMB had its notorious ‘Spineless Soiree’ (otherwise known as the invertebrate ball) today. We all dressed up as our favorite marine invertebrates and strutted down a catwalk in the middle of our dining hall. I went as the purple urchin, S. purpuratus, along with Renee and Megan. We all carefully crafted urchin spines out of purple wrapping paper and huddled together with our spines in all directions. We also brought a little lantern to represent Aristotle's lantern. Although it was hot, messy, and pretty ridiculous, it was still a lot of fun!
I’ve had quite an exciting week in lab; the time finally came to test out some of my probes! I tried out the two Ect2 mutants I was making last week that have mutations in the regions that are thought to bind Rock (affectionately termed Putative Binding Regions, or PBR). In one version of the mutant only the Rock binding regions are mutated (simply PBR), but I also created a version of the mutant that contains both the Rock mutation and the Snowflake mutation I documented in week 3 of this blog (Snowflake-PBR). I started injecting oocytes with both versions of the probe alongside wild-type Ect2 and Snowflake Ect2 controls. Left: a bat star with a protruding stomach (a sign of a healthy animal and ripe ovaries!) Right: a starfish without a protruding stomach. Unfortunately, the oocytes I have injected so far have been largely uncooperative. They did not express either probe at a high enough level to image, and many of them seemed quite sickly. The ones injected with the PBR probe also had odd zones of bright green Rho activity and behaved very strangely when I added hormone. Despite this oddity, they had dim Ect2 expression, which was a little discouraging. The snowflake-PBR mutants also had somewhat dim expression, but I have hope that the batch of oocytes is at fault, not the batch of Ect2 mutants. One of our dissecting microscopes that we use to sort embryos and look at eggs, larvae, jellyfish, and lots of other cool things! We will be taking this microscope with us to the Charleston Marine Life Center to educate the public about our research. Aside from injecting and imaging a number of oocytes this week, I have been getting ready for presenting my project at the Charleston Marine Life Center this weekend. I fertilized some of my bat star eggs, so hopefully I will have some larvae by Saturday that little kids can look at under a dissecting microscope. I am looking forward to talking to the general public about my research, but a little concerned that my research is difficult to explain to kids. Hopefully they will appreciate looking at movies and larvae as much as I do!
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AuthorI am a rising junior at Carleton College, majoring in biology with minors in Neuroscience and Russian. I'm very excited to be working in Dr. George von Dassow's lab this summer, where I will be studying cell biology and embryology of marine invertebrates. Archives
August 2019
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