Living in “Ecological Time”

With the arrival of all of the interns and the start of the children’s camp this past Tuesday, Aspen T.R.E.E can really begin to settle into a routine, and I can begin to understand how this internship, and consequently my research, is beginning to take shape. Arriving by 9 A.M. at Aspen T.R.E.E allows the interns to have an hour of class time before the campers arrive. The subjects covered in class so far include the principles of permaculture, the development of edible forest gardens, and, as something not explicitly covered but I have an obvious interest in, ideas that help expand ecological orientations. I will expound on this more later.

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It’s a Marathon, Not a Sprint

The past two weeks of research for me have been all about paint. Not yet the cleaning of, but rather the initial drying process that oil paint undergoes immediately after being created. Oil paint has two distinct drying phases the first being within a week or so of being laid on a surface in which a rapid change in its T2 values can be observed. This was the main focus of my work this week, making paint and then running a series of CPMG pulse sequence measurements at 20 um increment depths to create a profile of the paint and its signal decay and t2 values.

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First Post!

I realize that I am late to the “first post” party, but I guarantee you, I have been busy at work! Along with my current project, I have undertaken another, more grande assignment, so trying to balance the syntheses and biological applications of both projects have been tedious, yet fulfilling. For those who don’t know, my research is in the synthesis and application of unnatural amino acids (UAAs). My current project L-phenylalanine-4′-azobenzene (AzoBen) has been going somewhat smoothly. Since I have already created this UAA, I have been testing its abilities. The majority of my time spent testing AzoBen has been spent with my new friend Florence the Fluorimeter. A fluorimeter measures excitation and emission spectra, and since my UAA is photosensitive, I can understand how different wavelengths of light change the functionality of proteins with AzoBen. My protein of choice is Green Fluorescent Protein (GFP) which derives from the jellyfish  Aequorea victoria, and naturally glows. I have been comparing the wild type, or normal GFP, with GFP with AzoBen inserted into the 151 residue, and also with AzoBen inserted into the 66 residue. Through the data given by Florence, I was able to note several interesting changes in emission spectra. When GFP was irradiated with light (365nm) the emission graphs would both shift up and shift down, and well as shift left at certain excitation wavelengths. This indeed means that AzoBen’s photoisomerable ability is affecting this protein, so this validates my project! While this is exciting, much more fluorimetry data needs to had before I can fully comprehend the specific effects.

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Waiting for my cells to divide…

Re-entry into lab work has proven to be an interesting challenge. I arrived on campus this past Sunday and have been analysing data from CAT ELISAs performed during the semester and performing plasmid preps to stockpile plasmids for use this summer. This break between the end of the school year and the beginning of the Summer Research Term is the longest I’ve had since I entered college; my catharsis regarding breaks is that it’s amazing how little time it takes to fall out of good study and work habits. I am still in the process of regaining or attempting to regain the same level of focus I had during the semester and have found that the transition to a more studious approach does not occur with the same ease that flicking a switch does, however, it’s a relief to be able to come back and devote time to research, rather than splitting time between research and classes which always happens during the semester. The concentration that studying and working requires also brings a sense of quietude and serenity that is unparalleled in any other tasks. The first week back to research passed quickly and was consumed mostly with administrative tasks and planning aside from data processing and plasmid prepping. Plasmid prep brought up some pertinent questions for future transfections. When the prep yields high concentration samples, should they be diluted down, requiring a higher plasmid transfection volume and increasing the likelihood that the plasmid will disperse throughout the transfected plates, or should the concentration be kept at a high level and lower volume? Plasmid dilution may introduce impurities while smaller volumes of higher concentration plasmid may not disperse throughout a transfected plate as well as the higher volume, lower concentration plasmids. I will be performing a CAT ELISA this week to determine if the data from previous ELISAs was valid and this will hopefully give me a point of comparison to determine whether or not plasmid dilution is worth the risk of impurity introduction or if this step in the process even effects a significant difference in the ELISA results. If the variability in results from the past ELISAs can be lowered over the course of the summer, I plan to move onto CAT ELISAs with Thyroid Hormone Receptor Beta (I’m currently only working with Thyroid Hormone Receptor Alpha to minimize the chance of error introduction which can occur when working with larger samples). The purpose of the CAT ELISA is to quantify the amount of CAT Enzyme expressed in transfected cells. Experimental cells are transfected with both a Thyroid Hormone Response Element (TRE) and the suspected exportin; the TRE is necessary as it is the site of Thyroid Hormone Receptor binding. Thyroid Hormone Receptor can bind to the TRE regardless of the presence of Thyroid Hormone (T3), however T3 presence generally allows the receptor-hormone complex to act as a transcriptional activator. As a transcriptional activator, transfection sets are performed as duplicates with T3 added to one of the duplicates, allowing comparison with T3 deficient samples. If the protein acts as an exportin, data would show decreased levels of CAT Enzyme expression relative to cells that were not transfected with the protein. Analysis of these transfected cell lysates after performing the CAT ELISA protocol and comparison with control cell lysates will enable characterization of the protein as an exportin or demonstrate its deficiency in this area of function. The long break between the end of last semester and the beginning of summer research has also meant needing to cultivate more cells to work with and unfortunately, cell growth doesn’t just occur when one dangles colchicine over a flask and threatens the cells to divide, it involves waiting for the cells to divide and reach the proper confluency. Until then, I’ll be updating my lab notebook, reviewing literature and protocols, and getting to know every cracked spine and bent page in my MCAT books (and hopefully next week I can forget all of these features while transfecting and performing ELISAs).