Lab Session 1- Summer Progress

Working in a research lab is overwhelming, frustrating, and draining. I do something wrong nearly every day, spend hours repeating the same complicated procedures, and almost never achieve my desired results. Despite all that, these past two weeks have been an irreplaceable introduction to what undergraduate research really is. I learn something new each day, and get closer to being self-sufficient in the lab setting with every new procedure.

Going into this summer, I was under the impression that I would be continuing work on the Glaser-Hay dimerizations project discussed in my last post. I have found, however, that the Young lab has many projects running throughout the summer. In this post, I will briefly outline which projects I have been working on as well as what progress I have made in each department.

 

My main project for the summer involves protein arginine methyltransferases (PRMTs), which control epigenetic expression through the methylation of arginine residues on chromosomes. This methylation determines which areas of the gene can be accessed by transcription machinery, which in turn determines which sections are expressed. A small change in the amino acid monomer structure of these proteins can change its function as well, leading to a difference in epigenetic control. My research aims to make use of this property by controlling epigenetic expression through the use of PRMTs.

In order to activate or inactivate PRMT by changing its structure, I need to integrate an unnatural amino acid (UAA) into the structure of the protein. This UAA is othro nitrobenzyl tyrosine (ONBY), and contains a caging group. When the caging group is removed through the decaging process, ONBY can the become ‘activated’ (and subsequently activate the entire protein) through irradiation by UV light (365nm).

Through the process of bacterial transformation, I have created two sets of cells: one with the ability to express the PRMT protein in its wild type form (single transformation, PRMT WT), and one with the ability to express PRMT protein that can successfully integrate my UAA during protein expression (PRMT-TAG + pEVOL-ONBY). After growing the cells on a plate, I made starter and expression cultures for both PRMT wild type and PRMT-TAG + pEVOL-ONBY. To test whether by ONBY was successfully integrated into the protein, I irradiated a small sample of PRMT expressed with ONBY to determine if UV light could make the inactive protein active again. Both proteins were run on a gel to determine purity and yield.

So far on this project, I have successfully expressed PRMT wild type, PRMT-TAG, and GFP-66 with ONBY. I have also made (mostly) pure ONBY, and I will continue on working towards greater yields of the UAA. Although my first ONBY synthesis was not able to reactivate the protein (I suspect an issue arose during the decaging process), more syntheses of ONBY should result in successful irradiation. This project has been a great bridge between the spheres of biological and chemical research, as I spend just as much time performing biological procedures (cell transformation, protein purification, etc.) as chemical (UAA synthesis, decaging, etc.).

 

The second project that I have been working on – with a fellow student – is similar to the Glaser-Hay project discussed in my previous post. I am continuing work with the Glaser-Hay reaction, but with different components involved in the synthesis reaction. I have used bipyridine, bipyridine in carboxilic acid, tris(2-carboxyethyl)phosphine (TCEP), and several other reagents in an attempt to find the combination with the highest yield of bioorthogonal conjugations. I have also run time trials to determine what reaction time works best to avoid protein degradation. Each trial is run on a gel to determine yield.

Beyond these two projects, I also assist other students in the lab with their projects whenever I have the chance. I have learned how to use all the equipment in the lab, as well as how to make or restock the reagents used around the lab. I have learned an immense amount about the research process in a very short span of time, and hope to continue that trend through the second summer session.

Comments

  1. semodlin says:

    Greetings! I’ve heard great things about the Young lab and it sounds like you’re having a blast (and also it’s a lot of work, but it’s still fun!). I have some questions for your project just because I’m a curious person:
    1. Do you know what purposes Arginine does for chromosomes? Is it just used as controlling transcription?
    2. Do you use just GFP? My lab uses both GFP and mCherry just to make sure our transformations work

    Cheers

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