MARIN ACADEMY RESEARCH COLLABORATIVE​
STUDENT BLOGS
Update 3/17/19
This week I am going to begin experimentation for my genetic cross. I am going to begin the process of making a wt male stock tomorrow, and after that I will be able to begin with my two different crosses.
I have been in contact with Dr. Marc Hammarlund, a researcher at the Yale School of Medicine. I reached out to him after reading one of his papers about the use of RNAi feeding when targeting neuronal genes, which has been proven to be less effective than RNAi feeding in other genes. He has given me some guidance on the issue of RNAi feeding vs. genetic cross. I am still not completely decided between the two, but I am going to begin making a male stock in case I go in the direction of the cross.
Update 3/10/19
This week I will be finalizing my plans for my genetic manipulation. I have scheduled a call with Dr. Maureen Berg, a researcher at UC Berkeley who is going to help me talk through my plans for either a genetic cross or an RNAi feeding. I have also sent out an email to researchers at Yale who have posted studies about RNAi feeding in neurons, hoping that they can give me some advice on my procedure. Regardless, I am hoping to finish up the planning this week so that I can jump into data collection.
I picked up new cultures of the wt and glod-4 strains at the Buck institute, so I will be ready to begin genetic work once the planning is worked out.
Effects of increased serotonin and dopamine levels on lifespan of glyoxalase impaired and overexpressed Caenorhabditis elegans
This project aims to study the effects of glyoxalase impairment and overexpression on serotonin and dopamine looking at the glod-4 gene. The hypothesis is that glod-4 inhibited worms will show decreased levels of serotonin and dopamine, and glod-4 overexpressed worms will show either no change or slightly increased levels of the neurotransmitters. After treatment with exogenous serotonin and dopamine, I expect to see lifespan rescue in all three strains.
Update 2/18/19
Although I was hoping to begin the genetic cross last week, unfortunately I am not there yet. I am instead in the middle of trying to organize a meeting with researchers at the Dillin Lab at UC Berkeley to get help with the cross procedure. I also still need to pick up new stocks of the wt and glod-4 strains from the Buck Institute to use in the cross, and I need to order the dopamine mutant. However, as of right now, the only thing standing in the way of those goals is coordinating with mentors, so once the planning is worked out, I should be ready to go.
Update 2/11/19
The experimental plan has been finalized, I just need to gather my materials and get more information about the process for crossing and genotyping. I am going to look at just dopamine, not serotonin, using a locomotion assay and lifespan assay to compare the phenotypes of the glod-4 and GFP worms, as well as the glod-4 and GFP when crossed with dopamine mutants (restricted and over expressed)
Update 2/4/19
In the past week, I have slightly changed my experimental plan. Instead of using exogenous drug treatments to manipulate serotonin and dopamine, I am now going to perform a genetic cross with a serotonin or dopamine mutant. I am most likely going to pick either serotonin or dopamine to focus on, not both, but I’m not sure yet which one will be easier to do in terms of the assays. I am going to spend the next week gathering supplies and planning the genetic cross, with the goal of starting it next week.
Update
1/28/19
This past week, Rachel and I organized and started analyzing the data from our first lifespan assay. We created a survival curve in Excel, and we found that all of our strains started dying earlier than they should have, and we aren’t sure why. It could be related to the use of FuDR on the plates. I am also still working on reading through papers and trying to decide what methods to use for the rest of my experiments. I am talking to researchers at the Berkeley Dillin Lab, as well as Dr. Sanjib Guha, who recently left the Buck Institute on Aging. I am hoping do finish planning my procedure and order all of my materials by some time next week.
Update January 21, 2019
After an extended winter break due to illness and surgery, I finally dove back into my project again last week. I concluded my initial lifespan assay before leaving for break, so I now have to average the data and create a viability curve to analyze the data and determine if it is sufficient.
I am hoping to begin conducting the egg-laying and ethanol-avoidance assays soon, but first I am working on figuring out what supplies to purchase, specifically which types and concentrations of neurotransmitters I should use. I am currently in contact with multiple experts who will be advising me on the methods and materials I should use, and then I will begin true data collection.
My other task for the coming weeks is to research and make decisions about the methods I will use for statistical data analysis. This should be done before I begin data collection, not after, as to limit bias in my analysis.
Quarter 2 Update
In the second quarter, I made a lot of progress in solidifying my experimental plan and starting the beginning stages of my research. I began my initial lifespan assay, which should finish up in the next week. I will then analyze the data, which will hopefully be sufficient to point to a clear conclusion. It’s also possible that, with the few worms that were lost throughout the assay, that there is not enough data and I will have to re-assay the initial lifespan. Hopefully, the data analysis will support my hypothesis that the glod-4 strain has a shorter lifespan than WT, and that WT has a shorter lifespan than GFP.
I also spend the second quarter perfecting methods for plate preparation and seeding and worm synchronization. I hadn’t performed many of these procedures since last year, so it was helpful to refresh and tweak certain pieces of the procedures. I also spent a lot of time reading literature to determine methods for the later pieces of my project, including the egg-laying and ethanol-avoidance assays. My plan for these procedures has still not been solidified, but I am aiming to know my procedures by the beginning of next semester.
The first semester in general was the time for refreshing, perfecting, and researching both previous procedures and my plan for the rest of my project. By the start of next semester, I will prepared to gather my materials and dive into the actual experimentation.
2018 SCHOOL YEAR PROGRESS
So far this year, I have continued to work on my experimental plan. I have continued correspondence with my mentors, including Dr. Sanjib Guha at the Buck Institute, and researchers at the Dillin Lab at UC Berkeley. I am trying to develop a specific methodology for my serotonin and dopamine assays, and I’m starting to organize my workspace and prepare materials for data collection.
GOALS: PLANNING
1/11/18
I am returning from a 3 week break from working on my project. My main focus for the next few weeks is to continue to narrow down and flesh out the details of my experimental plan. I need to continue to find more articles, read them thoroughly, and contact more experts to consult about my project.
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At this point I have a broad understanding of my topic, but I need to learn more about the procedure of engineering a virus. I need to decide what exactly I am going to engineer a virus to do, and then figure out how I can test the virus realistically and safely using the resources available.
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Another goal for the next few weeks is to revise and add detail to my research proposal. This involves thinking more about my general timeline for the next year and a half.
JDRF RESEARCH 101!
1/22/18
Tomorrow I am going to the JDRF conference in San Francisco. I think it will be a really valuable opportunity to learn about what is going on in the field right now and to get inspiration for my project. Sometimes journal articles can be difficult to digest and understand, so I am hoping that listening to presentations will be a more accessible way to learn about T1D.
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This past week I mostly focused on revising my research proposal draft. In the coming week, I want to wrap up work on my proposal, as well as reading more articles and doing research, hopefully with inspiration from the conference. I am also working on reaching out to more experts to continue to develop my project plan.
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1/28/18
​The JDRF Research 101 presentation last week was really interesting. Specifically, one interesting aspect of the presentation was the research that is being done on beta cell mass replacement, and techniques of enveloping new injected beta cells so that they aren’t threatened by autoimmune attacks. Also, it was interesting to learn about the technological side of T1D research, such as the development of the artificial pancreas. As this information relates to my project, I was intrigued by the possibility of engineering a virus that can help with the replacement of beta cells and inhibition the auto immune response. This week, I intend to use this specified angle of viewing my project to do more research into possible methods for my project. I am also going to continue to work on my research proposal and applications to summer programs and internships.
CREATING EXPERIMENTAL PLAN
3/9/18
These past few weeks I have been working hard at refining my focus for my project. I’ve shifted away from virus creation, and instead I’ve decided to look at bacterial systems in the gut microbiome of C. Elegans, a worm used as a model organism. While doing further research into the mechanisms of diabetes, I discovered studies describing the link between serotonin and the glucagon pathway. By focusing on how to regulate glucagon, I’m hoping to find alternate possible therapies for diabetes. I’ve developed a general plan for my project, which is to identify bacteria that produce serotonin, manipulate and overexpress this bacteria, and study the effects on glucagon levels. My goal for the next few weeks is to develop a specific experimental plan and methodology based on this outline.
I recently contacted the Kapahi lab at the Buck Institute to collaborate about their work on the gut microbiome of C. Elegans. This connection will also be an important part of planning in the next few weeks.
This week I am arranging to collect the dsRNA that I need and hopefully beginning my RNAi feeding process. I am talking to researchers at Berkeley and at the Buck who are both trying to help me locate the bacteria I need for the feeding, and once I pick it up, I will be ready to start data collection.