Category Archives: Week 4

We have proof of concept that our RNA trans-splicing technology works in vitro. We’ve shown that we can efficiently edit pre-mRNA by transfecting and transducing Human Embryonic Kidney cells (HEK293) and are planning to move into patient-derived cardiomyocytes (human heart cells) which we have differentiated from induced pluripotent stem cells (iPSCs). These two in vitro models, HEK293 and cardiomyocytes, provide a testing platform through which we can spend time increasing the efficiency of our proposed pre-mRNA editing tool. The goal is to increase efficiency as much as we possibly can before we go on and test the technology in humanized mouse models (in vitro). The current experiments we are running are done in a controlled environment and simply cannot accurately model the numerous cell types and molecules that are present in an actual organ. So we know that once we begin in vivo testing, the efficiency of our tool will drop in magnitude because of the many unpredictable variables that come into play in mouse models.

I felt this update was needed to give context to the things I do on a day-to-day or weekly basis. As I previously mentioned, our general aim is to increase the efficiency of our technology in vitro by running transfection and transduction experiments on HEK293 and differentiated cardiac cells. This means that I am in charge of keeping these cells alive and well so that we have plates of cells available to repeatedly run experiments on. The HEK293 cell lines are quite resilient and fairly easy to maintain, the stem cells are not. We cannot culture stem cells with antibiotics because it inhibits their proliferation and so we must be very careful to not contaminate them with bacteria and kill them. Whenever I deal with the stem cells I wear a lab coat, nitrile gloves, and protective sleeves. Also, 75% ethanol spray is a good friend of mine. You can never use too much ethanol spray to disinfect the items you work with within the cell culture hoods.

In addition to cell culture, I do transfections and transductions of the genetic material necessary for our technology to work. This means that I am responsible for introducing the technology into cells while also keeping them alive and well. A couple of days after introducing our tool into the cells, I am also responsible for extracting the RNA from each of the wells in the plates, synthesizing DNA from that RNA, running PCR amplification on the synthesized DNA, and then submitting that DNA for sequencing. This is roughly the entire pipeline, and the process of seeding cells all the way through submitting for sequencing can take 4 days in HEK293 cells and up to 19 days in our stem cells. The extra 15 days are included in the case of stem cells because that’s how long it can take to fully differentiate a place of stem cells into cardiomyocytes. On any given day, I could be working on any time point in this process. 

In the future, my work will also include working on a directed evolution model to have the power of molecular evolution aid us in finding our prime construct.

(Here’s a pic of me with my stem cell drip)

As one would expect, working in a tissue engineering lab revolves around one main thing: cells! In the lab there are myriads of ways to manipulate and care for cells and ensure that they are happy. In a sense, every day in the lab is like cell daycare where we have to cater to their every need. We also have to use procedures to analyze the cells and tissues that we culture to derive scientific insight from them.

Typically I arrive at the lab around 11 after getting a hearty breakfast from McDonalds and a fruit-flavored drink. As soon as I set my bag down, it’s go-time! Usually there will be cells in the incubator that I need to passage; a procedure which lifts them off the bottom of the flask and allows me to move them into 3 new flasks so that the cells have enough room to grow. I also might need to change their growth media, which just involves aspirating out the old media they’re in (which has accumulated cellular wastes) and replacing it with a new media, before putting the cells back in the incubator to grow. These are the most quintessential daily chores as we need cells in order to do research. There are also lots of side-projects that I will have to work on throughout the day. Usually there will be samples of cross-sections or whole bundles that I need to immunostain. This process sometimes takes 4 days, so if there’s a whole bundle stain I will start on Monday and replace them in blocking, Tuesday to add primaries, Wednesday to add secondaries, and Thursday to mount the samples to a slide. This process sometimes takes from 45 minutes to an hour, but is mainly washing steps which just require waiting. Sometimes I’ll also need to create cross-sections which involves putting a whole bundle in OCT (a compound which can be frozen quickly in liquid nitrogen with tissue embedded within). I then use the CryoStat to cut sections of the whole bundle and can immunostain these samples as well. Recently I’ve been working on practicing Tube Formation Assays, a process which allows one to evaluate endothelial vasculature formation. I’ve also been making molds by synthesizing a polymer so that we can grow whole bundles in them. Usually I will finish in the lab at 4:30 – 5:30, and may read a few papers to further my understanding afterwards.

My time in the Volkan Lab consists of primarily two procedures, PCR/running gels and ChIP (chromatin immunoprecipitation). This means that my day is usually split into half collecting/dissecting fruit flies and pipetting for these two procedures. I usually start my day by collecting Ir84a mutant male flies (we only study gene regulation in males so they have to be separated from the females) and then preparing buffers so that I can start ChIP by moving onto dissecting previously collected males. Depending on how many flies were previously collected, I will either do an antenna or a head dissection which take roughly 90 and 30 minutes respectively. Once the dissections are done I proceed on with the next steps of ChIP over a course of a couple days. These steps include fixing, washing, homogenizing/sonicating, immunoprecipitation and qPCR. This process of ChIP is the primary tool being used to test the hypothesis of my project: Ir84a mutants will have the chromatin wound more around the fruitless gene than wild-type flies.

In addition to ChIP, I also make and run a lot of gels. The primary purpose for which I run the gels is to confirm that the lines of flies I am using are indeed mutants for the receptors in question. For example, I am studying flies that are mutants for the Ir84a receptor and so I perform PCR on these DNA samples with  primers for the Ir84a gene. If a band is seen on the gel for this Ir84a primer, then the line was not actually a mutant line, but if no band is seen, then the line is indeed a mutant.

My first stop on my way into the lab is my little lab bench/office space to drop off my backpack. Then, I always go to the shared office space (where there is usually fresh food someone brought) to check in with my bench mentor, Julia. She and I go over the plan for the day, and we both write it down on a sticky note to keep track of our “to-do list.” The actual details of the plan vary from day to day, but, most often, it includes me returning to my bench to first prepare some PCR samples and sticking them in the thermocycler. Then comes the waiting.

There tends to be a lot of inactive time in the lab when Julia and I discuss the project, next steps, what’s actually happening while we’re waiting, etc., but usually this time gives me the opportunity to update my lab notebook. I spend a lot of time organizing it, taping in important pictures of gels we’ve run, writing down data and results and next steps. As someone who likes to keep a physical planner and bullet journal on hand for organizational purposes, the physical lab notebook is the perfect way for me to stay organized, and I make sure that it is kept pristine and well-detailed.

About 30 minutes before the PCR is done, I head over to the main lab space to pour a gel to run with the PCR products so we can image the gel and see if the products are the right size. This doesn’t take too long, and usually by the time I’m done, the PCR is nearly ready. After the samples are ready, I take them over to the shared lab space to add the loading buffer and wait for the gel to finish setting up. Carefully, I load the samples to a finished gel sitting in a buffer solution, then I hit start and let the gel run for about a half hour. This waiting is more anxious, because we’re so close to seeing if our reactions worked. Then it’s time to image the gel, print out pictures, and Julia and I discuss what the next step is.

For the first few weeks, it was often more PCR and more gel-running, but now we are getting to an even more exciting part of our project where I’ll get to learn new procedures, and hopefully perform a biolistics transformation within the week. Even though I’ve grown a lot in terms of becoming adept at performing many of the day-to-day lab procedures on my own, every day is a new adventure in the lab, and each hour is an opportunity for me to learn something exciting.

While every day has a different to-do list, two tasks remain consistent: collections and dissections. Every morning, we check our boxes of fly vials for the ones we need to collect from. With tons of crosses going at the same time, it’s hard to keep track. To collect the flies, we dump the vials onto porous pads that emit carbon dioxide to put the flies to sleep. Then, we can look at them under a microscope to see if they have our desired phenotype, genotype, and age! This process allows us to have flies with everything we need in their genotype by crossing lines that already exist. For example, with this method we can create lines with a mutated spastin gene, but only in their neurons! We can also make lines to control for the technology that we’re using; if the technology was causing a phenotype, then that could mean our spastin mutation isn’t causing the results we see. 

Aside from collections, we also dissect larva of interest. Because mutants with deletions in the spastin gene have bunched terminal boutons and tiny synapses, dissection is key for our lab to study different types of spastin mutants. Dissecting consists of placing a larva in a dish with pins in it. Using forceps, pins, a saline solution, a microscope, patience, and expensive tiny scissors, we pin the larva down and create “fillets”. We fix these fillets so that they remain stable while they go through several washes, solutions, and antibodies. Finally, we mount the fillets onto a slide so that we can look at them under the microscope with fluorescence. Recently, we learned how to “score” a slide, which means we count the number of terminal boutons on a particular muscle. Difficulties arise when boutons synapse on different planes of focus on the microscope, meaning they aren’t synapsing on the same muscle even though they seem to be in the same area. Despite having difficulties with scoring, I’m beginning to develop an eye for finding muscle 4 and counting the terminal boutons that we are interested in! 

In addition to collections and dissections, our days consist of working out crosses on the white board, talking with Dr. Sherwood about the science, or going through papers. We also recently started a PCR project to determine if some lines actually have spastin deleted from their genome. Even though it’s been a struggle at times, Shibani and I have learned a lot and have developed our skills in collection, dissection, paper reading, scoring, washing, staining, and more!

Shibani and I preparing potential spastin mutants for PCR

A typical day at the Wright lab usually starts early in the morning. I get dressed in long sleeves, thick pants, and hiking boots and meet up with the two graduate students I am working with who pick me up around 8:00 am. We then drive a few hours until we reach the particular site we are surveying that day. We have been visiting various locations where the pitcher plant Sarracenia purpurea are found. Depending on the site we visit, we may spend a few hours walking through longleaf pine savannas searching for these plants, or we may just take a short walk along a path and find several plants fairly easily. We often drive to multiple areas within the same sites, taking gps coordinates of the plants we find. Once we have thoroughly searched that day’s site for purpurea, we drive back to Durham where I am dropped off at campus. I then head to my dorm where I make sure to check for ticks.

For the past several weeks, we have only been surveying the sites we’ve visited. However, we will soon return to these sites, where we will take fluid and leaf samples of these plants, which we will later study and analyze in the lab.

On the days I don’t do field work, I usually read papers that cover a broad range of topics, from Sarracenia purpurea morphology, to the organisms that are found in the pitcher’s fluid, to concepts of food web ecology and how they apply to these plants.

In the Chilkoti lab, we work to create different proteins that could potentially improve drug delivery. To do this, bacteria must be constantly transformed, grown, and lysed. Each step takes hours, and depends on whether or not the previous step was successful. Because of this unpredictability, workdays can only be planned one or two days before. While each day may seem similar, every step is important for making a functional protein we can store and use for future purposes. 

Some days we start at the beginning, with different pieces of plasmids we have to recombine. After cutting the plasmids using certain types of enzymes, we have a new piece of DNA that can be transformed into bacteria using a heat shock. These bacteria now have new plasmids that contain our desired protein sequence of ELP, and we allow them to create the protein as usual overnight or after a day. This step can take many hours, but necessary to allow for a greater yield of protein.

After a large amount of bacteria have grown, the protein must be extracted by lysing the cells. This erupts the membrane and allows the protein to be released into the surrounding solution. Afterwards, the desired protein must be separated and purified from other contaminants, which can be done by utilizing the ELP’s change in solubility under different temperatures. By switching between hot and cold temperatures and centrifuging the solution, the ELP can be isolated from other proteins that are always either soluble or insoluble. Through these cycles, the ELP becomes purified and ready to use for further testing.

It is important to note that these steps can be unsuccessful, and so a large amount of time is also devoted to checking our work and making sure we can continue with the purification. Without running gels to check DNA or protein size, we can’t be sure if the results we find are accurate. Although frustrating at times, the payoff of finding our sample to be purely our desired protein can make the days-long process worth it.

Tadross lab is known for its novel drug delivery system called DART (Drugs Acutely Restricted by Tethering) that allows delivery of conventional small-molecule drugs to specific cell sub-types. It is based on a covalent interaction between HaloTag protein and a HaloTag ligand that acts as a homing device.  This homing device guides drugs attached to the ligand towards neurons that express the protein. DART’s ability to be specified to a single cell type allows for a more precise investigation of potential malfunctions in the circuitry involved in neurodegenerative diseases. For example, DART has been used to restrict the binding of an inhibitor to a “go” cell type allowing for the investigation of the circuitry of Parkinson’s disease.  Understanding the circuit mechanism of a drug enables the creation of more efficacious drugs for use in humans.

The overall goal of the project is to develop an orthogonal DART system, which would allow for two different drugs to be used at the same time. This system requires two unique pairs of HaloTag protein and ligand, where each ligand would only work with its specified protein. Tadross lab has a set of ligands with low affinity to the current protein. We are aiming to derive a unique variant of the high-affinity protein through directed protein mutagenesis, changing the amino acid sequence of HaloTag protein to have a more favorable binding with the second ligand. Creating a second DART protein will allow us to deliver two different drugs into two different cell types concurrently. The second DART protein will allow to manipulate brain circuits with even more precision and to develop combinatorial drugs as a treatment.

To achieve the development of the second HaloTag protein, Tadross lab developed a protein display system called GRIP (Gluing RNA to Its Protein). GRIP display creates a stable linkage between RNA and the protein it encodes. Using GRIP and a technique called Iterative Panning we can derive the best binding protein out of a trillion different protein variants. Panning for best binders can be compared to panning for gold, where more stringent filters of a pan dish extract the gold from the dirt. After each cycle, more waste is removed until only gold is left. Similarly, this is done with GRIP display, where protein variant mix is exposed to the ligand. Each cycle the weakest binders are removed, wheres the strongest binding proteins become a large portion of the overall mix. After a few cycles of panning, next-generation sequencing is used to read the resulting RNA and to determine the amino acid sequence of the protein. The goal after a few cycles is that the protein extracted is unique from the original DART and can be used in the orthogonal DART system.

Every day in my lab, like many of my colleagues, is a bit different. I start of the day with a meeting with my mentor Liz and then get to work on modeling in R. Basically, we can train the computer to recognize patterns based on comparing the complex system of matrices of different possible causal variables of social behavior variation (like sex, social “status”, and access to nutrition). From training the computer this way, we can see which factors really matter. Because these models have to run millions of times and take several hours to finish (and we have several models that we’re always adding to), I get these started early in the morning.

I also read usually at least one paper a day that Liz and I talk about at our afternoon meeting. Often, it’s about baboon social habits or life, but sometimes they talk more about complex statistical analyses. For my part, it’s a lot of googling random words (like oncogenic) and asking Liz what the heck different things mean.

I’ve only “gone in” once, because it’s so much easier to run models from Blackwell where I already have all my computer stuff set up, but getting a tour of the lab was a highlight of my time so far! The huge lab room was pretty cool, but I really thought the records room was interesting. It has paper records of the entire project going back to the 1970’s.

I’m really looking forward to this Friday’s in person (!!) lab meeting. Zoom meetings can be nice because they’re so convenient, but I really miss seeing people in real life and really won’t miss the awkward Zoom-caused silences and interruptions that will (hopefully) disappear soon.

I never thought I’d be handling rodents so closely, but lately, my life seems to revolve around them. As mentioned in my previous blog posts, my mentor and I are studying the impact of particular hydrogels on damaged stroke tissue in the brain. This requires administering stroke, hydrogel delivery, putting the mice down, and brain analysis. For the first step, you pick up the mouse by its tail and rest it on your palm to deliver it to a chamber that delivers anesthetics. Once the little one is under, you open up the head, administer the stroke, and drill a hole in the skull where you’ll put the hydrogel. A few days later, you inject the hydrogel, and then you can harvest brains at predetermined time points.

Being at the Segura lab, I’ve observed and performed procedures with both my mentor and other graduate students in the lab. I’ve committed to doing animal studies in the Segura lab, so if there’s nothing to do at the bench, I’ll go shadow someone in the surgery room. Friday, however, was the day the mice were sacrificed, and I watched my mentor perform the “brain harvesting” procedure and even attempted to help—but I’d need a lot more practice with handling surgical tools and trying not to snip organs that we need (which would suck). I’ve also done the brain sectioning at -20 °C and I swear I almost got frostbite (but I didn’t!). I can’t imagine doing surgeries on people, but I guess that’s why we have doctors for that.

I really enjoy working at the Segura lab. I love collaborating with undergrads and learning from graduating students, doing benchwork, and participating in mice surgeries! I can see myself doing this for the next 8 years, so maybe a Ph.D. is somewhere down the line? I guess we’ll see!

My first brain sections: very subpar, but we all start somewhere!

A day in the life of the Bilbo lab usually has some routine tasks that I do every day and then usually I learn a new lab procedure. For instance, in the morning I fill syringes full of ethanol for the drinking in the dark experiment (DID) we’ll do by 3 later in the day. Then I check on the mice’s water bottles, and every week I get to weigh them. After these things get done, I usually find Julia, my mentor, and I get to learn how to do a different protocol that she’s using for one of the projects she’s working on. For instance, over the past week, we’ve been running behavioral tests on the mice we’ve been working with for our DID experiment. The first test we did was an elevated plus-maze. This is a way to quantify the anxiety response in mice. The maze is in the shape of a cross, two arms have walls that enclose them and the other two are open. We put a mouse in the center then record what they do. The more time spent in the enclosed arms, the more anxious the mice are.

photo from: https://www.e-phy-science.com/services/behavioral-tests/elevated-plus-maze/

Next, we did another test called the Barnes maze. This maze is a circular structure that has holes around the edges. One of the holes is an escape hole that the mouse goes into. We designate which hole we want that to be, then we place the mouse in the center of the circle and record how much time it takes them to find the hole. They then get to do this again 3 times. Now, this maze tests the mice’s memory because you conduct the test again the next day. Using the same escape hole, we use the same procedure, and hopefully, the mice remember where the hole was from yesterday. On the third day you switch it up, now the only thing you change is where the escape hole is. Typically, the mice go to the previous escape hole first because that’s what they remember and then it takes them some time to figure out where the new escape hole is. Since we have been running behavioral tests, the mice haven’t been drinking during their dark cycle, but usually, we give some of the mice ethanol and record how much they drink over 2 hours 3 days a week and 4 hours on Thursday. I’ve even gotten to run DID by myself. After recording the data in Julia’s lab notebook, I plug in the previous week’s data into excel and then plug the values into some statistical software that creates different graphs. 

image from: https://conductscience.com/maze/portfolio/barnes-maze/

I like that on a day-to-day basis, I’ve settled into a routine with some of my tasks and I feel pretty confident about doing them by myself. But I also enjoy how almost every day, I’m learning how to do something new, and I feel like at the end of the program I will have gained a lot of new skills.

Every morning this summer I take the bus and walk to French Family Science Center and report to my lab, Hargrove lab. I usually walk into the office before the lab itself, where I drop off my backpack in my designated workspace (I have my own one!) and greet all the other members of my lab. I then always meet with my mentor, and I show her any data I analyzed the night before and we have a discussion about where we should proceed next in our experimentation. Now that it is week 4, these discussions are very interesting and my understanding has improved so much that my mentor really values my input!

Next, I proceed to take out all the materials I need for the assays I am going to run. We keep everything in freezers to help preservation, so I always have to brace myself as my hands reach into -20 degrees Celsius freezers and even -80 degrees!

Once I take everything out and let it thaw, the chemistry begins! I usually begin by taking my RNA samples (which vary depending on what assay I am running that day) and nanodrop them. Nanodropping gives me a value of purity for the RNA (RNA purity changes on the daily basis!) which I use to make calculations on how much of each reagent I need to make the solutions I will need that day.

Once I have made all my calculations, I prepare the solutions. This can take a while, as I often need to heat up and ice the RNA solutions. Once the solutions are all made, its time to pipette!

As I am testing whether small molecules bind to specific RNA targets or not, there are usually 2 things that must be added to wells in a microplate for every assay. First, the small molecules are added. I sometimes do this manually, and other times my mentor uses a robot in the medical center to do this. Next, the RNA solutions are added. I always do this manually, and have become faster as time goes on. No matter how fast I get though, this is quite the process. Sometimes I pipette into over 2,000 wells in a day!

Once everything has been added to the microplates, I spin the plates in a centrifuge and leave them sitting for a while to let the reaction play out so that it reaches equilibrium.

Finally, I read the plates on a machine that detects fluorescence. The plates are read one by one, and I always save the data on a flash drive so that I can analyze it later.

Example of a microplate that I prepared.

Me in the lab

Life in the lab varies from day to day, but most consist of at least one of the following: observing or doing surgery, slicing / imaging / recording from visual cortex (V1), or figuring out Matlab.  Performing surgeries on mice has definitely been the most surreal part of working in a lab.  There have been multiple times where I stop and ask myself “who let me do this?”  That said, it has also been the coolest part.  So far, I’ve learned how to do two surgeries (burr hole injections and perfusions) but I’ve done significantly more burr holes than perfusions.  Although I’ve only performed those two types of surgeries, I’ve observed others in the lab doing other kinds of surgery.  Everyone I’ve shadowed has been great in answering any questions I have about the surgery, and overall it’s been incredibly rewarding to witness.  So far, I’ve mainly done practice surgeries and have been injecting dye into V1 instead of the actual virus.  However, last week we began injecting the virus.  This is exciting because it means that we’re going to be able to start collecting data over the next couple of weeks.

We collect our data by slicing the brain and either imaging the slices or recording cellular activity. With the practice dye injections, I’ve only imaged under a microscope.  We were able to see where I injected the dye (and whether or not it was where I was supposed to inject it).  We got decent results, and it was nice to get some validation that the surgeries I was doing worked.  I’ve also enjoyed learning to use all the equipment because it makes me feel like I can be more independent.  Once we finish up the virus injections, and wait a couple of weeks to get it to express, we’ll begin recording from live cells.  I’m hopeful that we’ll see good results.

Finally, Matlab is the program we use to do our analysis.  For this project, we use it to create graphs of cellular activity (that we’ll collect from the recordings) across different conditions.  Given that I have zero prior coding experience, there is definitely a learning curve that I haven’t quite reached yet.  However, once I become more comfortable with Matlab, it will be cool to see our results and be able to tell whether our experiments worked.  

Overall, my time in the lab has had a healthy amount of variety.  It has been a good balance of building a repertoire of skills while focusing on perfecting the ones that are particularly relevant to the work I’m doing.  It’s crazy to think that I’m working towards the point of being able to carry out experiments with minimal help.  I love that I’ve been able to learn so much in such little time and that every day brings new opportunities to learn even more!