Happy Friday (or should we say, Fri-YAY) from CauseScience!
psgurel– Today I am miniprepping! If you remember last week, I was doing PCR to get a specific DNA construct. After doing PCR, there are several steps before you have nice clean DNA. For the DNA I’m using (plasmid DNA) the final step is to extract your DNA from bacteria. Lucky for us, several companies make “miniprep” kits that make this process super quick and easy. It takes about 30min, and then you have (hopefully) nice, clean DNA!
crestwind24– This is crazy! I am also doing mini preps of DNA this morning!! SAMESIES!! Preparing DNA is a major part of most labs, as made obvious by todays post. I am making DNA that will label synapses in neurons in C. elegans. Once I have the DNA that I want, we will inject it into developing embryos, and then I will have transgenic worms!! Hopefully with glowing synapses!! This will allow me to visualize connections between different neurons.
CauseScience Friday… more like mini prep Friday!!!
crestwind24– Today I am doing a bunch of VERY serious molecular biology (as you can see from my picture)!! I am extracting DNA from C. elegans and genotyping them for a transgene that I hopefully added to their genome. In short, I performed an experiment to add my DNA of interest to the DNA of the worms, and now I need to check for worms where the addition of my DNA of interest worked. Cross your fingers for me! Maybe this serious face will change to a smiling face by the end of the day!!
pgurel– Today I’m preparing grids for cryo EM. I’m troubleshooting some problems (as I mentioned previously, I’m trying to develop a new method), and the first step of preparing grids is to make sure they are extra clean. One step I take is to clean grids using a glow-discharge plasma treatment. This is basically a fancy way to try and remove all impurities or contaminants from the grid surface. For highly sensitive techniques like cryo EM, even very small impurities (smaller than a speck of dust) can ruin the experiment, so it’s incredibly important to make sure grid surfaces are clean! Here I am with the plasma cleaner!
psgurel – I’ve just spent most of my morning in lab meeting, and will spend my afternoon going to a few lectures. Presenting work and getting feedback is a critical part of science. Lab meetings are somewhat more informal sessions where the entire lab can get together and discuss specific details on members’ projects. It’s helpful for all involved in terms of troubleshooting, fine tuning direction of the research project, etc. It’s fun, but can also be somewhat tiring. Here’s my post lab meeting selfie 🙂
crestwind24 – This morning I came into lab very early to make sure I got all of my experiments done before heading to Philadelphia in the afternoon. One of the things I needed to do was run 4 PCR reactions and then image the products of the reactions using gel electrophoresis. Or more simply, I needed to make specific fragments of DNA from the C. elegans genome, and then make sure I made the correct DNA by looking at its size. When I visualized the PCR reactions separated by size on an agarose gel (top panel), I did not see the size DNA I was making with the PCR. Instead I saw a bunch of randomly sized bands and smears (hence my grumpy face when sitting at the gel imager, bottom left). Luckily, since I was in lab so early, I redid the PCR at a different temperature and got nice crisp bands at the exact size I expected (right bottom panel)!!! I love when molecular biology works!!!
crestwind24- I am spending part of my day processing images (micrographs) that I took earlier on our labs confocal microscope. As I mentioned in a previous CauseScience Friday, the confocal allows me to take amazing pictures of neurons and their axons and dendrites. Today I am taking many images taken through the depth of a worm and making 3D animations. This allows you to see the morphology of the axons – or where they are in space. Below is a partial low-res GIF I made of one of my animations – it shows two neurons and their axons in C. elegans!!
psgurel- Part of joining a new lab involves developing a new project. Today, I’m doing some test runs as an initial step in developing a method for imaging different conformations of actin filaments for cryo Eelectron Microscopy. First, I have to coat EM grids in a mechanism that will allow the actin to bind properly, so I’m surveying different ways of coating EM grids. Wish me luck!
crestwind24– Today I am at an all day meeting in NYC, the New York Area Worm Meeting! Basically, the meeting consists of discussions and presentations from labs that study C. elegans in New York City and the surrounding area! Very excited for a nice day of research talks downtown at NYU!! Followed by sushi with my co-workers this evening!!
psgurel– Getting my hands wet with my first round of experiments in a new lab! I will be doing a lot of structural biology, specifically trying to determine high resolution structures of cytoskeletal elements. One technique I’ll be using in the new lab is Cryo Electron Microscopy. This is different from negative stain EM (as mentioned on a previous CauseScience Friday); the method of preparing samples is different and much trickier: samples are flash frozen in liquid ethane (which is liquid at -180C… COLD) so they maintain their natural/native state. This gives us a much better idea of how these proteins ACTUALLY look. Today I am imaging samples that have already prepared the day before!
crestwind24- This morning I am working on my lab’s new confocal microscope (middle). Using this fluorescence microscope, I can take high resolution images of neurons in C. elegans, including their amazing axons and dendrites!! To do this, I use worms that express fluorescent markers in distinct neurons. The fluorescent proteins then fill the neuron cell bodies, and can be excited with lasers to emit light!! I then use the confocals photo multiplier tubes to detect the light and create an image! The image on the right shows 2 neurons and their processes (one red and one green)!
psgurel– I have been spending this morning packing and getting ready for the American Society for Cell Biology (ASCB) Annual Meeting in Philadelphia! This is a 5 day long international conference where thousands of scientists, exhibitors, and students gather to discuss science, network, learn about career opportunities, and more. I’m pretty excited because this year I will be giving a small talk on my research, and I will be participating in a panel on science advocacy. It’s my 5th time attending this Annual Meeting, and I’ll be posting and tweeting throughout the conference.
psgurel – I’ve spent most of this week turning in my thesis and preparing for my defense next week. But today, I’m taking a break from all of that and repeating an experiment! Oftentimes, we must repeat experiments to prove that we are confident in our results. While it may not be thrilling to do the same experiment again, it is awesome when a result is reproducible! Today, I’m doing a similar kinetic experiment as I did on Sept 19th. Woohoo!
crestwind24 – Today one of the graduate students in my lab is defending her PhD. I love going to PhD talks, it is always fun and interesting to see the story that has developed with the training of a new scientist!! Last night and this morning I spent a bunch of time baking a cake for her in the form of a C. elegans. It is a chocolate cake with cream cheese pudding frosting! The green dots and lines represent the two neurons that she has studied in her thesis! Good Luck and Congrats to Dr. Pat!!
psgurel: Today I am looking at actin filaments using Total Internal Reflection Fluorescence (TIRF) microscopy. You may have heard about total internal reflection when learning about optic fibers… basically, if you shine light at a particular angle, instead of going through a sample, it’ll bend back, or get “internally reflected.” The TIRF microscope relies on this principle and as a result, images acquired with TIRF have really good signal to noise ratio, meaning there is good contrast and low background. Our department recently got a grant accepted to buy this scope, and so today is my first day using this system. Before then, I had to travel to places like Chicago, Montreal, and Woods Hole to use a proper ‘scope. Anyway, check out our new rig, and check out what actin looks like after it’s been polymerized for an hour (looks like hair!).
crestwind24: Today I am doing some computer work, specifically I am looking through data from the modENCODE project! The modENCODE project, which stands for Model Organism ENCyclopedia of DNA Elements, is designed ‘to identify all of the sequence-based functional elements in the Caenorhabditis elegans and Drosophila melanogaster genomes.’ The project is working to identify all of the DNA locations where regulatory proteins or transcription factors bind DNA. Using the data from the project, I can analyze where in the C. elegans genome the protein I study is working. It is an awesome tool to complement the work I do in lab! For more info, check out the wikipedia page on the related ENCODE project, which is mapping the functional DNA elements in the human genome!
crestwind24 – This morning I am screening through many, many plates of worms. Usually this is very tedious, but today it is very exciting because I am looking for worms that have had their genomes edited using the CRISPR/Cas9 system!!! The CRISPR system allows scientists to make specific genetic changes in the actual genes of bacteria, cells, and animals. In the case of C. elegans, the CRISPR system allows scientists to target genes within the genome to mutate them, change them, delete them, or add things to them. It is extremely powerful, and hopefully I will find some worms that have the genetic edit that I designed!! Cross your fingers for me!
psgurel– Speaking of tedious, I am in the final steps of expressing and purifying protein. Purified protein is a requirement for a variety of biochemical experiments (like the kinetic assay or analytical ultracentrifugation I mentioned earlier). This whole process takes 5 days for the protein I’m expressing, where I’ve had to: grow and express my protein in E. Coli (E. Coli is typically used for protein expression because they grow very fast and yield a lot of protein), lyse the bacterial cells, harvest the protein through various methods of chromatography, concentrate my protein, then store it appropriately. Here I am posing with the FPLC, in the final step of purifying my protein. Looks like I got a good yield, yippee!
psgurel: I always save my favorite experiments for Fridays! Today, I’m doing an assay to look at kinetics. ATP (Adenosine triphosphate) is typically known as the main “energy” source in cells and is required for several reactions to take place. On a chemical level in these reactions, ATP gets hydrolyzed and you are left with ADP (Adenosine diphosphate) and phosphate as a product. Today, I’m looking at how fast different proteins hydrolyze ATP. To do this, I stop reactions at various time points, and then I add a green dye that labels free phosphate. The dye turns darker shades of green as the amount of phosphate in solution increases. Check out my samples! The darker the green, the more phosphate is present, which means more ATP has been hydrolyzed!
crestwind24: Today I am scoring and imaging the neurons of old worms. This means sitting for a few hours in a dark room at a big microscope and computer. Specifically, I am trying to see what is happening with certain neurons in the worm as they grow old. We know very little about how neurons change in old age, and why some die in normal aging, as well as in many diseases. In the first picture you can see the green light under the microscope, which is activating my fluorescent proteins. On the computer screen you can see the image the microscope is taking. The arrows point to the 2 worms I was taking picture of, old worm selfie!! The selfies are a bit grainy since the room is dark… sorry … and its time to upgrade my phone.