Author Archives: Demi Zhu

The End

This summer has been a really enjoyable experience, and time has flown by so quickly. It feels like just yesterday that I moved into my apartment in 2015 Yearby, nervous about starting my first day of research and worrying about screwing up the experiment. I had worked in the SJR lab during the year as a research assistant autoclaving tubes and making media, so it was nice to actually be involved in research there and learn what research involves on a day to day basis. Although I’m a neuroscience major, I’ve always been fascinated by genetics, so a yeast genetics lab fits perfectly with my interest.

Besides enjoying my time as a researcher, I’ve also learned a lot and worked very hard. I didn’t expect to find out that coming into lab on the weekends is not a rare occurrence, especially when yeast forms a colony in 2 days. I’ve realized that as much as failure is daunting, it’s something that happens in lab whether you made a mistake or not. Results aren’t always what you expect to happen either – and even though you might want it to be a mistake, you double check and confirm that those results are right. Furthermore, I’ve seen example after example that really do prove that working hard is the only way to achieve your goals, and I plan to continue working hard in the future.

While I know I’ll have to work hard in the future, I’ve also been thinking about my career path. I’m strongly considering getting an MD/PhD, not just an MD or a PhD by itself. I didn’t expect to like research as much as I did, but I’m happy that things turned out this way. I can’t wait to continue researching in the fall!

(PS A huge thank you to Dr. G, Jason, and the Trinity College for giving me this opportunity this summer, I really appreciate it!)

Choices

These past few weeks, I’ve truly enjoyed hearing all of the faculty seminars. I love learning about their research and how they had different backgrounds and paths yet all ended up here at Duke. In terms of research, it probably comes as no surprise that I really enjoyed Dr. Williams’ talk on aerobic fitness and neurogenesis because I love neuroscience, and I’m probably going to major in it. I actually took Neuro101 with her as my professor, and it was interesting learning about the research she does. Normally, when you think of exercise, you think of your muscles getting stronger, from your arms and legs to your core. However, normally people don’t think about exercise affecting their brain. Dr. Williams’ research shows that exercise/aerobic capacity does in fact affect the brain. A higher aerobic capacity and more exercise leads to increased neuron proliferation or survival in different parts of the brain. Now I’m starting to regret all those times this summer I’ve debated whether to go to the gym or stay in and watch Netflix and chosen Netflix.

Beyond my choices this summer on whether or not to exercise, there’s also many choices that I’ll face in the future in regards to my career path. There isn’t one particular talk that stood out to me regarding their career path, but that’s only because they have all shown me how diverse the path can be and how it can change. From being the director of the Duke Lemur Center to working in industry or academia to receiving dual degrees, everyone eventually ended up doing what they love. Of course, the road there isn’t easy and takes a lot of hard work, but I’m ready to put in that hard work and hopefully, end up doing what I love too.

Many Yeast Colonies Later…

As the summer has progressed, so has my research. We got our first set of sequencing data back last week, and are hopefully going to get our next set back next week. The mutation rates have also been calculated, and there’s another set to be run in order to confirm the mutation rates for a couple strains. All in all, my summer project is close to being completed, we already have usable data that already narrowing our hypotheses down (but I’ll save what it is telling us for the poster session in two weeks).

In terms of the highs and the lows of research, I’ve learned that not everything will turn out like it should, and that even if you try not to make a mistake sometimes it happens anyways. In the sequencing data we got back, there was some noise, which was normal – you just delete the insignificant data and organize the data into a readable format. However, we tagged the different DNA with different barcoded primers, and primers were showing up that I did not put in to the PCR (maybe when I diluted some of the primers it could have contaminated the other wells if I was not careful enough). Thankfully, this was okay because I could match up the sequences that had a barcode that didn’t belong with sequences that did, which allowed the deletion of the out-of-place barcoded data. There was also a mutation that showed up in every sequence of one isolate of one yeast strain, which was probably a mutation in the original isolate, which was out of my control.

Overall, my research has gone pretty smoothly. It has, however, shown me that you can never be too careful, and you also need to be aware and be able to notice things that can help you troubleshoot the experiment.

Chalk Talk Reflection

I thoroughly enjoyed listening to everyone’s chalk talks last week. I had spoken briefly with some people about their projects and read about everyone’s projects in the blogs, but listening to the chalk talks really clarified my understanding of the research everyone is doing. I also love the diversity of the research – when I told my friend I was doing a biological science research fellowship, he automatically assumed I was doing cancer research, which is a very narrow view of biological research (though I’m sure he was half-joking).

Out of all the research projects, Casey’s research project on mast cells stuck out to me because one of my close friends is extremely allergic to nuts. (I went on a sort-of-tangent here but I moved that down to the bottom of this post in case you want to read that tangent*). I never thought about the mechanism of anaphylaxis before, but the fact that the granules in mast cells cause cell death in endothelial cells if ingested surprised me – it seems counterintuitive that endothelial cells would ingest them if it would cause death (then again, allergies seem counterintuitive in general – though I d remember reading an article a couple years ago about research into why allergies may a good thing**). I’m really interested in hearing the results on her experiment, especially because she revealed that in the first step of the experiment, the endothelial cells did die when exposed to granules from the RBLs. I can definitely see how this research could go on in the long term and be used to develop drugs that can help fight against an allergic reaction. I can’t wait for the poster session to see the results of not only this research but also the research of my peers in this program.

 

*Tangent: I was on an airplane with her once when the flight attendants began passing out peanuts (which is a thing I didn’t think happened anymore, I only remember being served pretzels on flights these days, but it’s highly likely that I’m just sleeping through the flight attendants passing out food). She covered her nose with her shirt because she was worried about breathing in the allergen, and I was worried she would go into anaphylactic shock for a good while before I relaxed. Once we were also trying to find a place to eat in a mall for a good twenty minutes before settling on the Cheesecake Factory because so many places in that mall cook with peanut oil or couldn’t guarantee no nut contamination.

**I can’t remember where this article was from but if I find it I’ll post the link here.

A Day as a Yeast Geneticist

I always start my day with coffee. After waking up in the morning and before arriving to lab, I drink a cup of coffee to prepare myself for the day. Once in lab, I go to my bench and create a to-do list to make sure I don’t miss anything that needs to be done (shout out to Jonny for that tip). After that, I get to work.

Depending on the day and what point I am at in the project, I do different things.

I might streak out yeast strains for single colony purification.

I might be picking individual colonies of yeast to grow overnight for a genomic prep or for a fluctuation assay.

I might do a follow a yeast transformation protocol to knock out a specific gene.

I might use prong plating to isolate mutants, and then replica plate them onto selection plates to get individual colonies.

I might do a genomic prep to isolate the DNA in order to use it in a PCR.

I might run a PCR to amplify DNA for sequencing or to check that the gene I want to knock out really has been knocked out. I’ll make sure I’m signed up to use on of the machines, grab a bucket of ice and defrost the things I need for the master mix. Then I combine everything and put 25 or 15 microliters (depending on what the PCR is for) into each well of the PCR plate.

I might run an electrophoresis gel to confirm the PCR.

I might purify the PCR product using a Thermo Scientific PCR Purification Kit.

I might combine the DNA (marked with a sort of “bar code” of DNA during the PCR) and measure the DNA contents before walking to the Biological Sciences building to send it off for sequencing.

I might be plating cells for fluctuations assays, or counting hundreds of individual colonies.

Of course, there’s the not-so-glamorous side of research too. Some days I might autoclave test tubes that I need to run my overnight cultures, or make and pour a few liters of a specific plate that I need to use.

All in all, I just try to make efficient use of my time and do what needs to be done. When the sequencing results get back, I’ll be able to start analyzing the data!

Mutagenesis and Yeast

The Jinks-Robertson Lab at Duke investigates genome stability and mutagenesis using yeast. Saccharomyces cerevisiae is used as a model organism because yeast is easy to grow, and has a cell cycle similar to that of human cells which is also regulated by homologous proteins. The project I am working on deals with mutagenesis in these yeast.

In wild type yeast at the endogenous location of CAN1, it has been found that there is a 2 to 1 strand bias for C to T mutations on the transcribed strand, which are reported as G to A mutations. When DNA mismatch repair is removed by knocking out the MSH6 gene, the strand bias is increased to 9 to 1. Thus, the goal of my project is to try and determine what is driving the mutations bias on the transcribed strand. Over the summer, I will be working with two possible hypotheses: that either 1) the transcribed strand bias is caused by a polymerase that induces mutations during replication or 2) there is a cytosine deaminase that is either more active on the transcribed strand during replication orientation or that there is a preferred deaminase consensus sequence on the transcribed strand.

To test these hypotheses, CAN1 was relocated to ARS306 in both replication orientations. 96 CAN1 mutants will be sequenced from each orientation, and the mutation rates will also be calculated through fluctuations assays. This will also be done in mismatch repair deficient strains, which will be constructed by knocking out MSH6 with KanMX.

Cytosine deamination is the spontaneous loss of an amine group, which converts cytosine to uracil. In humans, the enzyme AID facilitates cytosine deamination, but AID is not present in yeast. This deamination can be repaired, but when not repaired, uracil functions as a normal base, and it is templated by DNA polymerase as Thymine, which creates a C to T mutation. When sequenced, we read this as a G to A mutation. A possible cytosine deaminase that could be causing this transcribed strand bias in yeast is FCY1. Previously this enzyme has been shown to deaminate RNA, but it has not been shown to act on DNA yet. To test this possibility, FCY1 will be knocked out in both orientations using NAT, and the mutants will be isolated using prong plating. 96 of each of these mutants will also be sequenced and the mutation rates will also be calculated through fluctuation assays. This will also be done in FCY1 and mismatch repair deficient strains, which will be constructed by knocking out MSH6 with KanMX.

IMG_5932

Some selection plates for the FCY1 knock outs (a PCR is also run to determine which yeast colonies are FCY1 deficient)

 

 

We’ve already sent out PCR amplified mutants for ARS306 CAN1 strains, and I’m working on prepping the mutants for the yeast with MSH6 knocked out and FCY1 knocked out. The double mutants with both MSH6  and FCY1 are growing, and I still have to do the fluctuation assays. I look forward to continuing this project and getting the results as the summer continues!

An Interview with Dr. Sue Jinks-Robertson

Dr. Sue Jinks-Robertson is a professor of molecular genetics and microbiology and a director in the Program in Cell and Molecular Biology. She received her undergraduate degree from Agnes Scott College, a small liberal arts women’s college, where she studied biology. From there, she went to the University of Wisconson in Madison where she received a PhD in genetics. For her postdoc, Dr. Jinks-Robertson went to UChicago for 3 years, where she worked with Tom Petes on recombination. I always knew that the Jinks-Robertson lab and the Petes lab collaborated and were close, but I didn’t realize that the connection went back so far! People always say that networking is important to gain connections and build relationships, and this just shows how true that statement is. After her postdoc years, Dr. Jinks-Robertson worked at Emory for 20 years before moving to Duke in 2006 – after being recruited by none other than Dr. Petes.

When first starting out as a graduate student, Dr. Jinks-Robertson worked on bacteria. However, during this time, she became fascinated with yeast.In Dr. Pete’s lab at UChicago, she was assigned to work on recombination with yeast. She did not have experience with yeast at the time and thought she would be working with ribosomes, but she gave it her all and enjoyed the whole scientific process; after all, doing experiments is fun and like Dr. G says, it’s craftwork. “Yeast suits [her] temperament,” forming a colony in around 2 days, and while it takes a little bit longer than E. coli, yeast are more complex.

My dad, a physics professor, has always told me that he likes research a lot more than teaching, so I’m always interested in hearing what other people think. While Dr. Jinks-Robertson does like teaching, she also acknowledges how time spent teaching is time not spent in a lab, and there are only so many hours in a day. In her hours not spent in the lab, she enjoys spending time with family, working in the yard, and cooking.

To finish the interview, I asked her what advice she had for someone like me, who is just starting out in research and looking to become a better scientist. First and foremost, she said to do what excites you, because enjoying what you do is better than making a lot of money. When Dr. Jinks-Robertson was teaching genetics at Emory, a student who had a D in the class asked her for a recommendation letter for medical school. However, when she spoke with him, it turned out that his parents were the ones pushing him towards medical school, and he actually liked history the most. It seems like parents always want their kids to go to medical school, and I doubt that mindset will change anytime soon. Another piece of advice Dr. Jinks-Robertson has is to trust your instincts. I know I second guess myself often, so that is definitely something I have to work on.

I enjoyed talking to Dr. Jinks-Robertson during the interview, and I hope that one day I can be as successful as she is, doing what I love and maybe even bringing my dog with me to work (fingers crossed on that last part).

Week 1

“If you expect an experiment not to work, you’ll be pleasantly surprised when things work out.”

Though he did not use those words exactly, my mentor Jonny told me that when I was still a lab assistant making plates and sterilizing pipettes in the Jinks-Robertson lab. However, those words stuck with me, especially as Dr. G told us coming into BSURF that our project may not work. Jonny is naturally a sarcastic guy, but what he said encapsulates the fact that research isn’t like a lab in chemistry class where if you follow the instructions you’re sure to get an expected product. Things won’t always work out like you expect them to in scientific research, and if they do then you’ve lucked out. Thus, I don’t expect the project we are working on to be easy, but I do expect to gain valuable knowledge not only on lab techniques but also on what background knowledge has been used to formulate the experiment. I already feel like I’ve learned so much and it has only been one week.

Furthermore, I expect to make mistakes. Towards the middle of the week, I was working on a genomic prep of some yeast cells. One of the people in the lab was helping me with it, and towards the end of those steps for the day, we realized we had skipped a step in the protocol as she was teaching me. Thankfully, we were able to fix it because we still had products from the previous step. That really reassured me that it was okay to make mistakes, especially if even someone trained like her can accidentally skip a step, as long as we own up to them and find ways to fix them, even if it isn’t as easy of a fix as the one we encountered. It also reminded me not to be too comfortable in my knowledge, and to check off each step of a protocol and double check. Despite these precautions, I’m sure I’ll still make mistakes. We aren’t perfect, as much as we would like to be. I’ve just got to try and minimize the number I make!

This week has been a good one, and I’m excited to continue with research for the next 7 weeks!