Murphy Lab- Last Week

Richard again, continuing on from my last post.

My last week was a week with more experiments than usual.  On Monday, I did an STAM with the unc-73 mutant and wild type worms, and then tested chemotaxis up to two hours after training.  I analyzed the images I took of the chemotaxis plates on Tuesday, and bleached my egl-4::GFP worms with the hope of doing one last adaptation assay on Friday.  From what I observed, the unc-73 mutant did not make the food-odorant association as effectively as wild type, but it retained its memory better than wild type did, which is consistent with what Geneva found.

On Wednesday, I did the egl-4 and wild type STAM again, since there were various things that went wrong with my egl-4 strain in the past, and after analyzing my results, it seems that I have good data that confirms what is expected.  I did the goa-1 and wild type STAM on Thursday, and like the unc-73 mutant, the goa-1 worms didn't learn as effectively, but retained a longer lasting association between food and butanone.  Today I was hoping to do one last adaptation assay, but the worms that I had bleached were overgrown, and thus many had died, leaving only the young ones with very few eggs as the worms I had to bleach.  As a result, the worms grew very poorly, and I had to spend much of the day analyzing my data from Wednesday.

Overall, reflecting on these past nine weeks, I felt I've learned a lot.  Doing chemotaxis assays can get a bit routine, but it is nevertheless exciting to test new mutants and see what their learning and memory is compared to wild type.  I was really hoping I could test the egl-4 crh-1 double mutant, since no one else has done that so far, but sometimes matings don't work out.  Still, a lot of the STAMs I performed in the last few weeks were useful replicates for Geneva, and she is in the process of publishing her paper, which she said will most likely have me as one of the authors, since I produced some data that is of use.  And that's really cool, because not many high schoolers can say that they've been published.

Anyway I'd just like to thank Dr. Murphy for giving me the opportunity to work in her lab, Geneva for mentoring me all while she is dealing with her pregnancy, Dr. Peretz for helping me throughout the year, and everyone else involved.  Time to enjoy the last couple of hours I have left in lab.

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.

Glowing Worms -Last Week

Hi everyone, this is Rhea researching mitochondrial disease at CHOP.

Because last week, my life span results found that the gas-1 mutant worm had lost its main phenotype, a short lifespan, my PI asked my to perform florescence tests on it to see if it still had chemical differences  from the wild type. Since Fred, an undergrad, has been working with fluoresce for the past few months and perfecting the assay, he taught me what information fluorescence can uncover about the worms being studied. There are three different dyes that the worms are fed, Mito Sox, Mito Tracker Green (MTG), and TMRE. Mito Sox measures the oxidant burden in the animal's mitochondrion which should be higher in gas-1 mutant worms because the sicker animal experiences more oxidative stress in its cells. MTG measures the mito content in the animals cells and should be less in gas-1 worms because the mutant strain is born with less functioning mitochondria. TMRE measures the membrane potential in the animal's mitochondrial complex which should be less in gas-1 worms because their mitochondria are less capable of creating a concentration gradient with H+ ions.

To perform the fluorescence experiment, I first had to prepare enough plates. I had to spread them with OP50 e.coli and then with each dye. After placing about 60 young adults by hand on each plate, I incubated them for 24 hours so that they have time to ingest the e.coli with dye and the dye had time to adhere to the fatty cells in the animal. Then after 24 hours, I handpicked them again and transferred them onto a plate of fresh bacteria. This is done so that the worms can eat the e.coli without dye and clear their gut of the dye. We want only the dye that had penetrated into the cells to fluoresce not the food in the gut itself. After letting them clear their gut for 3-4 hours, I put levamisole (a drug that temporarily paralyzes them) on each plate making sure every worm is submerged in the drug. After giving the worms 30 minutes to fully paralyze, I took the plates to the microscope with a camera. I used different filters on the microscope for each dye, and zoomed into each individual worm and took pictures of each worm's pharyngal bulb.


Here's a good one:

About 2 hours of picture taking later I have to manually circle the pharyngal bulb using a software. This software then counts the pixels and measures the amount of light per pixel and calculates the necessary information. According to the fluorescence experiment, everything seems normal with the gas-1 strain. Nevertheless, my PI has ordered a fresh batch of worms for the lab to work with.  

Week 2 and 3: Reactors and Reacting

My name is Anna Piwowar, and I am currently working at Dr. Kartik Chandran's lab at Columbia University, working with ammonia oxidzing bacteria in batch reactors and studying their kinetics.

This week began with Medini and I cleaning out the reactors and setting them up for a new cycle. Between rinsing and autoclaving, it was a lengthy process. We had to overcome many problems (pieces that didn't fit, screws that had to be unscrewed, and everything in between). Finally, we thought we would be ready to inoculate our sparkling clean reactors and begin looking at cell growth. However, problems kept cropping up, and Murphy's Law held true: everything that could go wrong did. *(Not everything did go wrong, yet. So I don't want to jinx anything but we are still able to move forward with the process.) The DO (Dissolved Oxygen) probe on one of the reactors was found to be defunct, so now we are in the process of ordering a new one (and they do not come cheap). We do have the one reactor working properly enough for the time being, and hopefully we'll start collecting data on cell growth and nitrite formation.

While the process goes, I have a lot of time to read. Medini has many textbooks to offer me, and on Tuesday it was all about studying reactors, all the types and the equations. Sadly, I lack the Calculas to understand some of the things, but after an arduous tutoring session with Medini, I understand what the kinetics are and what we need and why.

By the end of the week we decided to go forward with the second reactor without the DO probe, and instead opting to manually find the dissolved oxygen. Even though we can't have simultaneous growth, we will at least have two sets of data.

The third week was all about data collection. We went forward with the second reactor, and now we're just working on maintaining the reactors and improving my laboratory skills. My PI is out a lot, but I will be meeting with him soon to talk about other things I might pursue for my last few weeks here.

Other than that, Columbia is beautiful, especially with the cooler weather, and having the chance to travel around Manhatten is wonderful.