Almost at the end…

 

A collage of pictures of me working in the lab, some of my earlier results, and me presenting those results at the ACS Senior Technical Meting

 

Oh Wow! How fast does time move around us….

I remember just as if it was yesterday, how I was writing my personal statement essay to apply for BioMinds. How it all revolved around the fact that I was interested IN A LOT OF DIFERENT FIELDS OF SCIENCE and couldn’t decide what to do with my life. Well in that essay I wrote down my wish for an experience that would help me realize what was that I wanted to do…

As it seems the fates were with me the day I sent out that application, for not only was I accepted for this wonderful experience; but I also got my wish. And funny enough what I wanna study now wasn’t part of the list that day.

From that list the one field that has stuck with my desire for the future has been Biochemistry, It is such a beautiful field, one that can explain everything that happens within a living organism based on ordered and highly regulated chemical reactions. But as my research developed I discovered a world I had never heard of, a scientific field that not only connects life science with chemistry and is a fundamental part of biochemistry, but it also involves physics and computer science. It’s as if it were a mix of everything wonderful in life. And its results are crafty and awe inspiring.

 

Protein crystals grown in space. Credit: Dr. Alex McPherson, University of California, Irvine. Source: NASA

I’m, of course, talking about Macromolecular Crystallography.

Now a days I work with fervor and passion with what I like most every Monday and Wednesday in Garriga’s Lab and I really see myself doing this for the rest of my life, so I’ve decided that once I graduate I will pursue a PhD in Macromolecular Crystallography focused on finding the structural information of proteins and relating it with their function.

 

My very belated second post of the semester…

“In your second post of the semester, describe the progress of your project and how far your results are away from your semester goals. Also state your achievements and the obstacles that you’ve had to deal with.”

Thus far in the semester, we’ve had a slow but steady progress. Weekly we process enough crude extract through Fast Protein Liquid Chromatography to have enough samples. And we also received the various reagents we needed, which mean I am ready to start crystallization screenings.  There are still no results though. So I am nowhere near my semester goals.  I don’t believe I’ve had any achievements and the only obstacle I’ve had to dealt with is not having enough sample which I’ve dealt with by being patient and following the isolation procedures over and over every week.

 

Prism 2011

As asked, this post will consist of the abstract I have submitted to the PRISM 2011 Conference.

Sanderson and Skelly state that macromolecular crystallography is the study of macromolecules (proteins and nucleic acids) using X-ray crystallographic techniques for determining their molecular structure. One of the main requirements for these initiatives is a high-throughput crystallization facility to speed-up the protein identification process. Thus, to determine the structure of a protein, the crystallization process is indeed very important. Egg-white lysozyme is a widely understood and researched protein which makes it a perfect model used to practice crystallization methods. Therefore, lysozyme was used as a model to learn these techniques. Hanging Drop Vapor Diffusion and Batch were used as main screening techniques. The screening conditions used for lysozyme crystal grow were a gradient of NaCl ranging from 5% to 30% w/v [ 5%, 7%, 10%, 15%, 25%, 30%] versus a gradient of protein concentration ranging from 25 to 80 mg/ml [ 25, 40, 60, 80] (for Hanging Drop) and three rates of NaCl|protein of 5.5%|50 mg/ml; 5.0%|55 mg/ml; 6.0%|60 mg/ml (for Batch), plus a NaC₂H₃O₂ 0.1M buffer to keep pH 4.8-5 to an optimum. It was observed that higher salt concentrations induce faster nucleation rates. Varying the NaCl saturation can alter nucleation process. Crystal growth was monitored for three weeks.

And so that you may have a good idea of what the presentation will be like, here is the poster I presented at the ACS Senior Technical Meeting.

 

Condition Screening of Egg-White Lysozyme Crystallization by Equilibrium Methods. Click to enlarge.

 

Having no progress can be very frustrating. But anyways …

We’ll, I guess I’ve pushed this dreadful post off, way too much. So here it goes.

For this, the last blog post of the semester, I am supposed to comment on my overall progress so far and the specific results obtained. Unfortunately my progress is non-existent.

 This semester I started very excited with the idea of starting the crystallization process of the Sulfhemoglobin complex, but after much consideration Ruiz and I decided to try crystallizing the Lucina pectinata’s Hb I- pCys complex instead. The pCys protein is an unknown protein that is obtained linked to Hb I when the last protein is purified. All we know about it is that it cannot be crystallized alone (the team tried exhaustively) and also that it is rich in Cysteine.

Given this I am going to try and crystallize the complex and when the crystal is obtained (if any are obtained) and analyzed we will predict the protein’s structure by subtracting the known Hb I structure to the crystallographic analysis. I will screen for conditions using the Hanging Drop Vapor Diffusion technique using four solutions that were previously tested by a colleague on summer. Two main precipitants will be tested: Ammonium Sulfate and Lithium Sulfate in different buffers and concentrations.

So far I have only practiced and mastered the different techniques and processes with the help of my immediate team, but this last Thursday I was finally told to design and submit my experimental approach and it was approved. This means that I will be starting tomorrow on the screening process.

Wish me luck.

Me extracting the ctenidia of Lucina pectinata, where the Hb's are at.

Surfing the blogs…

Once again we were asked to read some of our fellow BIOMIND’S bloggers posts and report on their investigations. My assigned blogs were Mayrim’s and Angela’s.

- Mayrim Bernard Vega, a UPR at Cayey Student, was a High School friend of mine and her project topic is:

“Structure-function studies of CC-chemokines: RANTES, MIP-1α, and MIP-1β”

Her investigation is about the structure of proteins. She has identified possible motifs in the structure of chemokines that can be similar to HIV motifs. She has also identified proteins that compete with HIV for the same protein-protein interaction motif and can suppress the interaction of the virus with cells receptors. To do this she used computer programs and bioinformatics. Now she is trying to create a pharmacophore model based on the proteins she identified (RANTES, MIP-1alpha and MIP-1beta). Also she will perform a drug database screening.

- Angela M. Alicea is a fifth year student at Rio Piedras campus and her research topic is:

“Vertical Transfer of antibiotic resistance Without the Selective Pressure of Antibiotics”

Angela’s investigation is on microbial ecology and is genetically oriented. She worked on helping a grad student on her thesis by identifying tetracycline resistance genes in the internal bacterial flora of mice and in their progeny’s. Their goal was to establish the magnitude of vertical transfer of antibiotic resistance as a problem we should face. To accomplish this she used PCR to amplify about 5 genes. Now she is on the process of writing an article based on this work and starting a new project which has a different target: Human Babies. Also she will now use a different amplification technique the qPCR also known as real time PCR.

As always the experience was gratifying since it exposed me to the research that is being done right now and the knowledge that is being sought. I am very pleased to have read about my fellow mates’ success and that the scientific community counts with such great talents.

And The Challenge Begins…

For this Blog entry we were asked to comment on our summer research experiences and state this semesters plan (objectives, techniques, etc.).

As I wrote on my last entry I have been preparing the myoglobin-sulf complex under different pH and buffer conditions in order to better understand the process for my next step. And well that is what I did during my summer, I worked full time from June 21 until July 31^st on monitoring the so called q-bands that the complex shows in Ultraviolet-visible spectroscopy, trying to rule out this conditions as factors involved in the unusual but yet often reported band displacement (a characteristic band in 630nm is often reported to shift to various wavelengths including 550nm and 680nm).

As for this semester the plan is for me and Beatriz Andujar to use the techniques I learned with Josiris (she learned them with Darya Marchany) and implement them in crystallizing the sulfheme complex, the major challenge is that this complex is unstable and tends to deteriorate. Because of this the PhD student and Aguadilla Auxiliary Professor Carlos Ruiz Martinez will teach us what he calls a “sandwich” assembly for Hanging Drop Vapor Diffusion and Batch Crystallization, and he will also instruct us on how to treat the sulfheme with CN for stability.  Using these techniques and following Chatfield’s method for complex preparation we strive to test different buffers and precipitants to find optimal crystallization parameters for the complex. This semester objective is to fully understand and manipulate this techniques and at least finding a proper precipitant for the complex.

And so the first year ends…

For this entry, which I’m submitting extremely late, I was to visit three other blogs < Keren Vallentin’s, Jessenia Flecha’s, and Andina Anton’s> and comment on the experience. Also I was to describe my overall experience this semester. Well about visiting other blogs I have got to say that it is very interesting and what science investigation is really about, being able to discover and share that knowledge. For what would anyone put so much time and effort if not to enrich as many as possible. I have got to say that every entry was interesting and invaluable, but the most appealing to me were Keren Valentin’s entries, because I can relate to her investigation.

She is dedicating her efforts to developing a biosensor out of a modified Au electrode and Hb I using Cyclic Voltammetry to identify and measure H₂S. Hydrogen Sulfide has various biological signaling functions but has to exist in a very specific amount in the body; a little less and one can suffer a number of mental diseases that include Alzheimer, too much and its toxicity can go from causing anemia and type I diabetes to being lethal; and thus the importance of such a sensor, which sadly does not exist yet. The sensors that exist nowadays either have a really short life span or don’t measure quantitatively, but their approach seems promising since Hb has such a high affinity for H₂S and the Au modifications seem to be working well.

Jessenia Flecha’s work is also something to what I can relate and understand thanks to the biotechnology program, she is working on creating a library of bacterial biodiversity on the Eutrophic Estuary System, Mandry Channel, in Humacao or so I understand. For this purpose she is amplifying the 16s rRNA (which is a very characteristic small sub unit of ribosomal RNA commonly used to identify bacteria because it does not mutate easily) through PCR in the hopes of sending her samples over to Rio Piedras’ Campus for sequencing. PCR is a very useful molecular biology technique that is leading genetics and genomics forward into a very bright future.

The third project, Adina Anton’s, is in exchange something I really don’t understand much, she is studying the effect of the transitional metals doping in electrical conductivity of ZnO, and measuring the effect of hydrogen in electrical collectivity of Pd. She wants to notice the sensitivity and the response time when the hydrogen it’s added, in order to know if the ZnO fibers and the Pd nanoshells can be used in the fabrication of hydrogen sensors. And even though I don’t know anything at all about material science or nanostructures, it is easy to follow what she is reporting because of the way she describes her project. This blog is the essence of scientific research, even though I didn’t know anything before reading it I could actually understand and inform myself.

About MY Research experience I have to say it took a rather pleasant turn, since my last post I haven’t worked much on crystallization because my mentor wanted me to understand the Sulf complex which is a big part of the projects within his lab and so I dedicated myself to test 3 different buffers and 5 different pH conditions for the formation of the Sulf complex with Myoglobin (Mb + O₂ + H₂S). This new approach is due to the fact that one of his graduate students is leaving and Beatriz Andujar (a fellow BioMinds and Lab Partner) and Me are gonna take over her project which is to crystalize this complex with Hb from lucina pectinata. This new project I will be engaging offers a whole new set of rules and challenges because even though I’ll still be part of the great team in the lab and will still have their support am basically now gonna be on my own (since I will no longer aid Josiris with her Thesis)  and secondly because this complex isn’t stable enough to be crystalized yet. The suggested approach my mentor gave me (used by Mariann J. Chatfield) is to use CN to stabilize it and that in itself presents a lot of challenges. But I will soon find out how truly I can handle myself, as soon as the semester starts to be more exact.