Adam Levine and Kelly Clarkson come to celebrate my last week at CHOP!! (kinda)

Overall my time at the Falk lab at CHOP this summer was really enjoyable. Though my stay was longer than expected, I got exposure to a number of techniques and had the opportunity to give a presentation to the entire genetics department at CHOP. It was definitely nerve racking at first, once got into my presentation it was very easy for me to talk about my project. Because I was going to present to  all the other PI's and lab members in the department I gave a few practice presentations to Dr. Falk. After spending a lot of effort of my powerpoint and doing a couple run throughs, I built my confidence. Rebecca and Fred, undergrads in my lab, also gave presentations on their projects. I really enjoyed my time at CHOP and also the commute through Philly since I feel much more comfortable with the city than before. Walking through CHOP everyday was also a humbling and eye-opening experience because I got to frequently see ill children and it reminded me of the difference CHOP makes in so many family's lives. On one of my last days at CHOP, Maroon 5's Adam Levine and Kelly Clarkson came to visit and I got to see them give an interview in the Ryan Seacrest Media Center. I managed to snap a picture too!!

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.  

Problems and Solutions (Week 6-7)

Hey guys! This is Rhea at CHOP, working on Mitochondrial Disease research.

Because I discovered that the mutated worms we're working with are showing less signs of well..being mutated, Julian asked me to preform a lifespan experiment without using FUDR to figure out if that was the cause of the strange results. FUDR is a chemical used to stop cell growth and this prevents the worms being observed to reproduce and infest our plates with hundreds of worm larvae. Julian has been using FUDR for many years now and it has never shown affects on the lifespan of worms but there has been a paper published which reads that FUDR may affect worms. I set up a lifespan experiment without FUDR to check if it was the source of lifespan longevity and when I checked on it the following day it looked like this.

The big clumps on the plate are colonies of contamination (bacteria). 

All of my lifespan plates looked like this and so I had to discard them all (about 25 plates of 20 individually picked and placed worms).

Because now I won't have time to restart my lifespan, I've decided to take on a fluoresce project which I've watched Fred do a few times. He grows worms in plates with different dyes which illuminate the pharyngeal bulb and he takes pictures of them under a special microscope. I'll be analyzing pictures to see if the mutated gas-1 strain compares to previous results to make sure the gas-1 are truly gas-1... addressing what I had found with my lifespan. 

The Real Deal Week 4-5

After a few weeks of waiting and disappointments, we're finally on a roll again. The last few weeks have been slow because we had a major contamination problem... meaning the plates we use to grow our worms had other bacteria and/or fungi growing in them. It took a while to pinpoint the source but after making batches of fresh plates, flasks of fresh E. coli and after taking extra precaution we finally have a stock of contamination free plates. However that was only the beginning of what’s to come…

The practice lifespan I started during my first few weeks in the lab is now coming to an end and the results show that the mutant worm is living much longer than it should be. The gas-1 strain (mutant strain) is supposed to have a median lifespan of about 8-10 days but my lifespan experiment shows that they are living 18-20 days…almost as long as the wild type (normal strain). Though I had begun prepping for my actual lifespan using drugs, my PI said that no experiments could be conducted with an animal that has lost its main phenotype. What she means is that the worm’s life span is a major phenotype that we use for experimentation and if this phenotype is not apparent in the animal we’re working with, then any experiments we do using this animal are worthless. All of the experiments in the lab that have  been working on has also been affected by this discovery. The mutant worm could be living longer than usual for a few reasons. 1. A random beneficial mutation may have occurred in one of the worms and then outlived the mutant worms and reproduced (in a sense survival of the fittest)

2. A wild type worm may have somehow crawled onto the mutant worm plate and then outlived the mutant worms (again survival of the fittest)

So long story short, my original drug lifespan has been put on hold until we find a solution. 

$3,600 Responsibility and Mock Life Span Experiment! -Week 2

(Rhea - CHOP - Mitochondrial Disease Lab)

Now that I am much more acquainted with the lab members and the ways in which the lab runs, I'm being given a lot more responsibility. After learning to pour plates and spread bacteria, Fred (an undergrad) tested my skills by asking me to pour and spread my own plates without supervision. Taking great precaution, I made sure to follow the protocol correctly (ex. didn't blow up the autoclave machine, didn't let the agar solidify in the flask I prepared it in). Fred meticulously checked my final products and informed me that my plates were completely contamination free (that's a huge deal!!). Now if the lab is ever running out of plates or solutions, I can be called upon to help them out. Just the other day during my free time, I made 2 liters of S. Basal, a salt solution used to maintain the worms, and 75 new plates for Zsoka to use. Later in the week, Julian taught me about the biochemistry work in the lab. We test carbon-13 labeled worms under different conditions for their organic acids, amino acids and protein abundance by using gas chromatography/ mass spectrometry and high performance liquid chromatography. To make the samples for each type of testing, the worms have to be grown synchronously (all at the same life stage) and under the specified conditions (ex. with drug, without drug), and then collected into viles. Once in the viles, they need to be centrifuged, ground up with a little drill, and treated with certain chemicals to ensure the best results. After showing me how to make samples with the first two viles, Julian left me to do the rest saying "you probably have a steadier hand and a better eye than me anyway" in his thick Russian accent. He also reminded me that every one of the samples cost us $150 to put through testing so I made sure to be careful with each and most importantly I made sure not to mix up the order of the samples...otherwise we wouldn't know which results corresponded to which sample! Overall my second and a half week in the lab has been even more exciting than the first and the best part is that my PI, Dr. Falk, told me that at this rate, I could be starting my actual life span project next week! Woo hoo!

My New Worm Pets! -Week 1

My first day at CHOP started out soggy and gloomy, but once I reached the tenth floor of the Abramson building and entered Dr. Falk's lab, I met two other friendly students working in Dr. Falk's lab for the summer (just to remind you I'm working in Dr. Falk's lab who works with Mitochondrial Disease at CHOP). Anyway, where was I? ...oh yeah so Fred, an undergrad at Penn, had been working in the lab during the school year as well and has a lot of experience with techniques and material in the lab. Rebecca, an undergrad at Notre Dame, very recently arrived at the lab and was in my shoes exactly a week ago. Before I could get my hands dirty in the lab, I had to learn the basics about mitochondria and about the subject of our experimentation, C. elegans. Essentially C. elegans is a 1 millimeter long, transparent worm which feeds on bacteria, typically E. coli. Fred told me that I will be working with two strains of C. elegans, N2 and Gas-1. The N2 strain is the wild type meaning it is the normal, healthy strain which has nothing wrong with it. The Gas-1 strain has a malfunctioning Complex 1 (a part of the electron transport chain in the inner membrane of the mitochondria). He also taught me how to pour agar plates (on which the worms and bacteria grow) and how to spread bacteria on the plates (the food for the worms) as I watched Rebecca do both. Fred also taught me how to use the dishwasher and the autoclave in case I ever ran out of clean glassware during my experiment. Then Julian, with whom I will doing my project since he is the life-span expert (the focus of my project), gave me my own worms to play with. He asked me to try to transfer them from plate to plate using his pick, which is essentially a rod with an extremely thin platinum wire at the end. From experience Julian told me that beginners usually kill their worms within a few days and tear holes in their agar plates but after a few hours of practicing I got the hang of it.

Here's a picture of my first ever plate:

The little lines you see are actually the ridges I made with the pick by accident when I tried to pick up and put down the worms, don't get too excited! The worms are much smaller and are very difficult to see with the naked eye...especially in this picture... sorry! Maybe if you zoom in?

My third plate:

You can see this one is much clearer and less damaged in comparison to my first plate. Woo hoo!! I made progress! I'm not a worm-killer anymore! The big blob in the middle is bacteria which the worms eat.

By the fourth day in, I have my own bench space, my own box of worms in the incubator with my name on it (yay!!), a lot more knowledge about C. elegans and the Philadelphia mass-transit system (phew!) and some awesome "colleagues" as Zsoka calls us :)

I hope everyone else at their lab is having fun and learning a lot too!

-Rhea