Annual Poster Day

I first have to mention that the Annual Poster Day was not well-organized. Some of the speakers were the same as in our first meeting. In addition, the vice-president of Amgen speech was exactly the same as the one he did at UPR-Cayey.

Then, we only had 30 minutes to present our posters. At least, a poster presentation should last for 1 hour. Moreover, there was a mess because the location was too small. Well, I tried my best to present and then to evaluate.

First, I talked with Juan C. Nieves and Suehelay Acevedo about their research. They worked encapsulating orchids to sell them as seeds. I think that their project was well-focused because they wanted to create those seeds to provide a better environment for orchids and allow them to grow. At this time, orchids are sold as adult plants and they are hard to maintain alive. In this sense, I think that their project could help to better the life of these plants by creating artificial seeds.

Then, I talked with Jaymie M. Tivera about an alternative treatment for cancer. She was studying tropical plant to extract a compound that could cure cancer. She said that the extract killed shrimps and that shrimps are similar to cancer cells (I sincerely don’t know why, neither her). In brief, I think that an alternative treatment for cancer would be excellent but I also think that this project is undeveloped and that she should read more about the project to be able to explain better.

Finally, I spoke with Laura Granda-Marulanda about the absorption of TNT from aqueous solutions with rubber based activated carbon. This project was well-focused because its aim was to treat polluted water with activated carbon obtained from old tires.

A new project…

At this time, all the Peg hydrogels have been polymerized and lysozyme crystallization is in progress. Because of this, I will grade me with a 3, in other words, with an intermediate progress for my experimentation. We are now waiting for the new crystals to form to obtain new XRD data and establish if the previous results are reliable.

In addition, during March, instead of working with different proteins -as I mentioned in the previous blog- I will be working in a ‘new project’. This project is related to what I have been doing during the last year, but I will implement a different technique with the Granada Crystallization Box.

The GCB was design to do four major experimentations: batch crystallization, crystallization inside a gel, crystallization inside a capillary while providing counter-diffusion with a precipitant agent inside the gel, and counter-diffusion adding the precipitant agent on top of the gel. The crystals that we will get for the new XRD were obtained with the last technique. In the other hand, during March I will be using agarose with the precipitant agent inside the gel as it was done by García-Ruiz, et al. The difference of my new project to the lattest, is that I will include the hydrogels inside the capillaries with the protein to enhance the crystallization process while simulating microgravity.

In general, the progress of my project has been constant and I have not had difficulties achieving my goals. Finally, below is the abstract of the poster that I will be presenting at the Annual Poster Day.

ABTRACT: Lysozyme Crystallization with Neutral Hydrogels Providing Conditions for Counter-Diffusion

Protein crystallization is of much interest due to its importance in describing and understanding a protein as it provides required crystals to perform analytical techniques such as X-ray Diffraction. Improvements in crystallization methods have provided the environment to crystallize some proteins. Counter-diffusion of a precipitating agent has been achieved by suppressing the convection and sedimentation by employing common polymers such as agarose. However, such polymers do not provide the means to control the diffusion coefficient of the precipitating agent and cannot be exposed to organic agents since they are not chemically bonded. This work focuses on the examination of the effects of poly(ethylene glycol) based morphologies on the crystallization of lysozyme. Lysozyme crystallization was achieved utilizing the counter-diffusion method by employing the Granada Crystallization Box (GCB). The precipitating agent was NaCl with acetic acid/sodium acetate buffer at 3.5M and pH of 4.6, respectively, and lysozyme concentration was kept at 40mg/mL. The crystals’ growth was studied by varying the morphologies of poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMA) and poly(ethylene glycol) dimethacrylate (PEGDMA). PEGMA (monomer) with MW of 200, 400, and 1000 g/mol and PEGDMA (cross-linker) of 400 and 1000 g/mol were used. These polymers were synthesized by free radical solution bulk polymerization. The polymer morphologies were modified by varying the monomer and cross-linker ratio. Results indicated that all morphologies were capable of producing crystals. The resulting crystals were examined by X-ray diffraction. Crystallographic results indicated that crystals obtained in the GCB are of lysozyme and that the crystals obtained with agarose, PEG 400/400, PEG 1000/1000, PEG 200/400, PEG 400/1000 had a mosaicity of 0.213, 0.216, 0.243, 0.279, and 1.268 degrees, respectively. These results indicate that cross-linked hydrogel matrices have the potential to provide diffusion controlled environments for crystallization and that this control depends on both, the monomer and the cross-linker. In addition, the crystals obtained with PEG 200/1000 were unstable and of low quality what implies that short monomers polymerized with long cross-linkers do not provide a well-controlled crystallization and vice-versa. This study will be repeated using other proteins and, as long term goal, proteins that have never being crystallized.

One semester to be graduate… a research to be concluded

 

During this semester, I mainly want to establish whether or not the XRD results that I have gotten are reliable. Because of this, I am polymerizing again all the PEG hydrogels and then I will crystallize lysozyme to see if the XRD results are the same. With this I will be able to conclude if the hydrogels are better than agarose to control counter-diffusion of the precipitant agent and, as a consequence, if hydrogels can optimize the crystallization process. A second goal for the semester is to work with proteins that are not as easy to crystallize as lysozyme to determine if we can get good-quality crystals of proteins different than lysozyme. Consequently, we have chosen to crystallize myoglobin and hemoglobin and then perform XRD to the obtained crystals and determine if the ‘optimized’ crystallization process provides good hemoglobin and myoglobin crystals.

As you may see, this semester I just want to repeat the experimentation that I have been doing and determine if the crystallization technique was optimized. Preliminary results show that we can be able to get good crystals with hydrogels but we do not have reliable conclusions yet. As soon as we get the conclusions that we need, I will move forward to use myoglobin and hemoglobin.

Since the experimentation is basically the same, probably I will not be working with new techniques. Nevertheless, I will continue using X-ray Diffraction which has been one of the most valuable techniques that I have learned. I used it on December to get quantitative, preliminary results. The technique consists of emitting x-rays across a protein crystal that will be diffracted by the electron contained in the protein’s atoms. The x-rays are then detected and a software generates a diffraction pattern that is then analyzed to determine how well organized are the atoms in the crystalline structure. If the atoms are well organized, the crystal is said to have good quality and this can help in generating an electron density map. That map then allows predicting the protein morphology which has excellent applications in the biomedical sciences such as: drug development, study of a protein behavior in a biological environment, etc.

Almost at the end of the semester… waiting to get last data from XRD to write a paper

In general, I have continued what I was doing last semester during these months. First of all, I achieved to polymerize the hydrogels inside the Granada Crystallization Boxes, which is excellent because a flaw in the experimentation was eliminated. That flaw was due to the manipulation of the hydrogels while getting them inside the GCBs. Now, that is not necessary because the gels are already inside the GCBs and contain an amount of precipitating agent. Since that amount of salt is not enough to reach super-saturation, some studies on the diffusion of a dye across the hydrogels were performed. With this, I was able to conclude that, in several days, the required precipitating agent concentration would be reached if an amount of the solution is put on top of the gel. Most important, I have many lysozyme crystals that would be analyzed with X-Ray Diffraction on December, to get some quantitative results that we have been waiting for during all the semester. And I wrote that we have been waiting for these results from Spain because most of my crystals were sent there on May but we do not have news on that. The purpose of this was to get data with the same diffractometer used by García-Ruiz et al when they publish the paper that reports the lowest mosaicity for lysozyme. As a consequence of not having results yet, I crystallized more lysozyme and I am ready to perform XRD at Río Piedras. Then, we will move forward to write a paper on this project. Furthermore, several days ago, I was speaking with my mentor and we decided to continue the same project with proteins that are harder to crystallize. The goal of this variation in the experimentation is to prove that our hydrogels control the precipitating agent diffusion in a way that will allow us to optimize protein crystallization in general.

In brief, this semester has been of much importance in my project especially because I had the opportunity to present it in ABRCMS and AIChE Student Conference.

In a week to ABRCMS and after that to AICHE Student Conference…

At this time, I am finishing my poster for the ABRCMS presentation and with it, I have noticed that my project have not progressed that much because the results of XRD have not been received from the people at Spain. They are supposed to be working with the same diffractometer mentioned in the papers related to my research.

Nevertheless, in a scale from 1-5, I would give my investigation a 3 because I have obtained, qualitatively, better crystals while polymerizing in glass Granada Crystallization Boxes. Though I have not got neither compared the quantitative data of the previous lysozyme crystals, which is in progress at Spain, with the data obtained during this semester, I have done the simulation mentioned in the previous posting. This simulation have confirmed that my results are consistent with what is expected.

One of the most important parts of my research is the analysis of crystals with an XRD to show if my lysozyme crystals have a better quality than those crystallized in microgravity at NASA. Hydrogels provide conditions of microgravity since they suppress sedimentation and convection effects. This is excellent to achieve crystallization but we need to know if the neutral hydrogels used in the GCB provide conditions for a diffusion of the precipitating agent that can generate a supersaturation profile adequate to crystallize good crystals. The major significance of this project is then obtaining good crystals of proteins that are actually hard to crystallize to perform them an XRD. This can make us able to study proteins that have not been studied in depth. Also, as long term goal, the project is directed to crystallizing proteins that have never been crystallized.

2nd Posting – 2nd Semester with Bio-MINDS

I have not learned a new technique yet but, as soon as possible, I will start working with a simulator of counter-diffusion inside a Granada Crystallization Box (GCB). This simulator will be very helpful in understanding the behavior of the precipitating agent inside the capillaries found inside the GCB

In a scale from 1-5 I would give the progress of my investigation a 3 because I have already polymerized inside the glass GCBs, I have crystallized lysozyme by employing the obtained gels. Nevertheless, I have not got neither compared the quantitative data of the previous lysozyme crystals, which is in progress at Spain, with the data obtained during this semester. As a consequence, I have not been able to really determine if the neutral hydrogels provide a better control of the diffusion coefficient than agar does. As may be expected, we have not started the redaction of a scientific paper since this is expected to begin at the end of the semester but I am very happy because my abstract was accepted to be presented at ABRCMS.

In achieving my goals I have not been able to polymerize hydrogels with the consistency needed to reduce the effect of gravity (sedimentation). Therefore, I have been varying the concentration of the precipitating agent since I think that the salt is not allowing the polymerization to progress as expected. This can be described, in general, as a trial and error process to determine the highest concentration of precipitating agent at which the polymerization can occur.

A new semester…

With the start of a new semester, new goals for my research come. Last semester, I was able to determine that different hydrogel morphologies controlled the diffusion coefficient of NaCl in different ways. This resulted on a variation of lysozyme crystals quality. Nevertheless, this study was performed in Granada Crystallization Boxes after getting the hydrogel in these small boxes. This means, that hydrogels were taken out of the polymerization container and transferred to the GCBs what resulted in an alteration of the real hydrogel “morphology” obtained via polymerization.

This situation brought me to a new main goal while performing experimentation. A way of polymerizing inside GCBs needs to be achieved. I have already tried to directly polymerize inside the GCBs but there is a problem, it seems that monomers react with the GCBs plastic what results on the broke of the plastic. Now, I am designing a new GCB, made of glass. During this week, the first trials with this glass GCB will be performed. If the polymerization is successful, I will be able to compare the controlled diffusion that each unaltered hydrogel produces.

In general, my goals for the semester will be:

• polymerize in the glass GCBs and crystallize lysozyme
• get an compare the quantitative data of the previous lysozyme crystals which is in progress at Spain (using the x-ray crystallographer that is used in similar published experimentations)
• determine if the neutral hydrogels provide a better control of the diffusion coefficient than agar does
• help my mentor in writing a scientific paper at the end of the semester

In brief, this semester’s research will be essentially to do the same as last semester but without altering the hydrogels to get a better understanding of the effect of them on the crystallization of lysozyme. Most importantly, we will try to get specific conclusions of the experimentation to write a paper, during the semester.

In addition, I will be developing my formal writing skills, specifically, while writing a scientific paper. For this, I will need to get and analyze data from x-ray crystallography. Other techniques or tasks to be employed could be the polymerization of hydrogels using ultraviolet light -instead of heat-, handcrafting (i.e., design and making a glass GCB), and the design of a new polymerization protocol depending on the results of the polymerization inside the glass GBC.

5th Posting

      During this semester, I have learned to do research since this is my first experience. I have also been doing oral presentations which contained literature review, protocols, and other new things for me. Taking this as a basis, I was able to create a poster which I presented on the Sigma Xi Poster Day. Furthermore, I have learned to prepare a laboratory notebook while investigating. By  other way, I have encountered some difficulties during the semester. Several times the experimentation was not successful but working it with patience helped me to complete all the objectives for the semester. Now that I have achieved my goals for the semester, I have also thought in my new objectives for next semester. Last week, I started with a Mesh Size Protocol which will help me to approximate the size of the polymer’s pores. This will be continued next semester in order to understand the effect of the hydrogels in the diffusion coefficient of the precipitating agent.

Laboratory skills

Some techniques that I have learned:1. free radical polymerization – this allows me to get hydrogels with different morphologies and study their effect while crystallizing lysozyme by counter-diffusion2. dialysis – introducing the hydrogels in membranes helps me to incorporate the precipitating solution to it and then crystallize the protein in capillaries by the diffusion of the precipitant (NaCl)3.  blanketing – this technique is very important for polymerization using free radicals; I use argon and nitrogen to take out the oxygen of the pre-polymeric solution to prevent it from reacting due to free radicals4.  counter-diffusion – this method is the basis of my investigation because I use it to crystallize lysozyme; to achieve counter-diffusion I suppress convection and sedimentation using hydrogels to permit the precipitant to diffuse in the capillaries and cause lysozyme to crystallize.

By other way, I have visited three bioblogs. It was interesting knowing a little of what other students are working on. Nevertheless, I would rather prefer to read and share information with someone which is working with something similar to my research. Below, are the blogs which I visited.

http://ilianemiranda.wordpress.com/ - selecting microorganisms that modify chemical complexes

http://biocam.wordpress.com/ - wireless endoscopy, optimization of the process

http://jctec.wordpress.com/ - creating an artificial nutrient reservoir for orchid embryos because they lack nutrients to survive

2nd posting

Group work… for us that is really easy because we are good friends. This is very important to achieve your goals in research because there is a lot of work to do and everyone has to complete their tasks. We, as friends, complete our tasks, first because we like to, and then because we think on our friends. If one of us does not complete our task the goal for the semester will not be achieved. In these sense, I have had to complete my experiments and, most important, understand them. For this, I have read several scientific papers related to the crystallization of lysozyme using the counter diffusion method. In brief, all of them consist of experiments using Granada Crystallization Box and agar to eliminate the convection and gravity effects while crystallizing proteins.