Murphy Lab- Weeks 7-8

Hi, this is Richard, and I'm studying learning and memory at Princeton.  I will break up the last three weeks I spent at the Murphy Lab into two posts, and this is the first of the two.

I spent the majority of my seventh week repeating the egl-4::GFP nuclear localization assays that I had started earlier.  Since we don't really know what is supposed to happen in regards to the GFP-tagged EGL-4 protein entering the nucleus, I wasn't able to make too much sense of my results for my naive and trained worms.  In some cases, the GFP localized into the nucleus of the AWC neuron, thus causing the nucleus to flash a bright green, and in other cases, it was mostly the surrounding cytoplasm that was lit up.  Such ambiguity held true in both the naive and trained worms.  We did however, know what to expect with my adaptation assay, so we tried to confirm this result in which for adapted worms, EGL-4 enters the nucleus and for mock adapted worms, it doesn't.  However, while in some of the worms the EGL-4 protein had clearly localized into the nucleus, in other cases it seemed that it was present throughout the entire cell, not just the nucleus, as evidenced by the entire neuron being bright.  Unfortunately, I didn't have enough time to repeat this again and get a definitive result.

I tried my egl-4 and crh-1 cross again, and got to the point where I had candidates who had a 1/16 chance of being double mutants, but I did not have the time to run a PCR and isolate the successful candidates that I could use for my double mutant chemotaxis assay.  I did a PCR earlier, but none of the 30 candidates I picked out were identified as double mutants, and within a 9 week timeframe, it's pretty difficult to start from scratch and set up the same cross again.  I was really looking forward to this, because, as of know, according to wormbase.org, pretty much the bible of C. elegans research, no one has tested egl-4 crh-1 double mutants.  I could've been the first.  But not anymore.

Anyway, I spent the rest of the two weeks on a couple of STAMs and an LTAM as well, to help with Geneva's project.  The difference between an STAM and an LTAM (long term associative memory) is that rather than training the worms once (starve, food w/butanone, test), I must train them seven times in 30 minute intervals (starve, train, starve, train...).  I didn't test chemotaxis, but Geneva used the worms I trained to observe fluorescence.

These two weeks were definitely more eventful than some I've had in the past.