Using liquid culture for the dauer assays has proven to be a little more tricky than expected. For whatever reason, we still haven’t been able to induce dauer even after drastically reducing the amount of food in the culture and doubling the ascaroside concentration found in the literature. Time to go back to the drawing board!
The leaving assays have been going well! Based on the data collected over the past couple weeks, we are going to hone in on a few of the sex reversal strains that looked promising. Over the final few weeks I’ll be working on collecting some more data and crunching numbers with Renee.
I can’t believe how this summer has flown by! In two short weeks we’ll be presenting our projects at a poster session- it seems unreal. I’m trying not to get sentimental just yet but as excited as I am to watch things come together over this final stretch, I can’t help wishing this experience wasn’t ending so soon.
Until next time! 🙂
Looks like our liquid culture protocol still needs some adjusting! First, the concentration of worms in the culture needs to be significantly increased to make plating them easier, so I’m hoping reducing the volume of the culture and increasing the amount of eggs added will solve this problem. Second, even with the small number of worms I was able to look at, there were hardly any dauer. Hannah thinks reducing the bacteria in the culture might help by preventing the response to the ascarosides from being reduced by the high availability of food. We really only need enough bacteria to keep the worms from arresting at L1. I’m going to give it a try and see if we get better results!
In the meantime I’ll be doing some trials for one of Renee’s experiments while she’s on vacation. Earlier on, did some mating assays for her which looked to see if male response to hermaphrodites or location of the vulva was impaired by any of the neuronal mutations she was looking at. This time around, I’ll be doing leaving assays which are similar in design. I’ll be working with tra-2 mutant males, which means they have pan-neural feminization. Any sex-specific roles of the neurons should be reversed, which may impact/reverse some sex-specific behaviors.When placed on a food source, males are known to be more likely to take excursions from the food or even leave it completely (presumably looking for a mate), where hermaphrodites are more likely to remain happily on the food. Tra-2 mutant males are known to have the leaving behavior of a hermaphrodite. I’ll be observing the leaving behavior of a few worms with additional mutations to see if any of these mutants will behave more like wild type males. This will help us take a look at which genes are involved in sex-specific leaving behavior.
Until next time! 🙂
So far, Hannah and I have been spending a lot of time trying to find an effective way to expose the worms to ascarosides. First, we tried adding the ascarosides to the plates before adding the egg prep. Hannah had previously tried mixing them into the agar, with no luck. Since then we have tried adding them on top of the agar and letting it dry, mixing them into the OP50 before we seeded the plates, and both at once. Still, none of the worms entered dauer. Now I’m in the midst of a different approach: liquid culture! There might be a couple of nice perks to using this method if it works out for me: I can easily get a large sample size, and I’ll know exactly the concentration of ascarosides the worms are exposed to. One minor downside is that liquid cultures can be more prone to contamination which would be a bummer, so I’ll have to be pretty careful about using sterile technique every step of the way.
I have the first batch on the shaker right now. We should know in a day or two if it is a success!
Here’s to hoping! 🙂
This summer I am picking up where Hannah left off on a project she worked on during her rotation here (with her guidance 🙂 ). I’ll be studying sex differences in entry and exit from dauer. For anyone who is unfamiliar with dauer, it is an alternative larval stage that young c. elegans enter in the face of certain stressors, such as high population density and limited food. Worms entering dauer undergo morphological and physiological changes that allow them to survive up to several months in poor conditions. Hopefully, I’ll also have the chance to look at the potential roles of three different pathways known to be involved in the regulation of dauer arrest: the guanalyl cyclase pathway, the TGFβ-like pathway, and the insulin-like pathway. I hope to be able to do this by inducing dauer arrest using identified components of dauer pheromone (a blend of ascarosides 2 and 3), and by observing entry and exit from dauer in him-5 and potentially daf-11, daf-7, and daf-2 single mutants.
Currently, we’re just working on adapting a procedure used by Srinivasan et al., 2008 in which they were able to induce dauer arrest using different combinations of ascarosides 2, 3, and 4 with varying success. So far, it looks like this will definitely take some trial and error!
Check back in a few days to hear about my newest successes (and otherwise)!
I’m Tylor, an undergrad who’s excited to be doing research in the Portman lab this summer! I study Biology at Siena College, and will graduate next Spring. Though I entered undergrad with the goal of going to med school, working in Dr. Adam Mason’s lab inspired me to consider grad school for research. This summer I hope to gain research experience and pick up some new skills, as well as form a better understanding of what grad school might be like and whether it’s the right path for me. I look forward to getting to know the other lab members and working on something a bit different from projects I’ve done in the past.
I’ll post updates on my project every so often, so feel free to check back to see what I’m up to!