Throughout my college experience in the Biominds program, I have obtained many knowledge and experience very important to my bachelor. These include many techniques such as: indirect extraction of DNA, titration (potentiometric or indicator), Polymerase Chain Reaction (PCR), extraction and lysis of cells, count plate unit (CPU), cultivate of microorganisms in plate (spread, streak and pour plate), purge solutions or solids, chromatography (partition, gas and paper chromatography), electrophoresis, electroelution, and electrogavimetric. Also, I am able to use equipment, such as: pH meters, Mel-temp, spectra UV-Vis, gel doc, among others things. This program help me to decide my future plans. My future plans are continue studying to obtain a PhD degree in Biochemistry. My main goal is to develop as a professional who offers all her knowledge, with effort and dedication, to help find solutions to problems that affect the environment that we live in and participate in Biominds program was mi first step.
Progress of the research project March 18, 2011
In the last semester I began with the research but I have not result. Therefore, in this semester we are optimizing all procedure to can obtain result. I am a student that put in the research all effort and dedication to achieve all goals. We start to extract and lysis of soil cells. However, the results obtained of these processes were not expected. In the soil sample have not DNA. We think that the manner of preparation of the solutions caused that the results were incorrect. This means that we cannot continue with research. In this moment we decide that must start newly with research. In this week we start to make all solution to extract and lysis procedure. In this opportunity we are start with research using the many accepts technique to no contaminate the sample. I hope that we can optimize the process and that can obtain the results expected.
Generation of Metagenomics Libraries from “Guánica” and “El Yunque” dwarf forests using an indirect method of DNA extraction. February 4, 2011
Abstract:
Metagenomics has become an emerging discipline which has provided culture independent strategies to access the more than 99% uncultivable microbes present in the soil. The expected diversity present, and the functionality detection in the sample, will depend on the DNA extraction method used. Previously, our laboratory generated several metagenomics libraries (Ml) with more than 800,000 clones from both forests in different sites using direct DNA extraction methods. Using these libraries, our lab. has been able to unravel activities such as antibiotic resistant and urease activity among others. While the Mls generated have been a valuable resource, no Ml has been generated on the proposed sites using indirect method. The main objective of this investigation is generating metagenomics libraries from the dwarf forests of the Dry forest in “Guánica” and the Tropical Rain forest of “El Yunque”, to further perform activity and diversity studies. The DNA was extracted from the soil samples using an indirect method where cells are embedded into agarose plugs, the cells are lysed in situ, the DNA fragmented, end repaired and ligated into a Fosmid. The last step requires the package of the fosmids DNA into a Lambda phages, and delivered the Fosmid into Escherichia coli Epi 300 by transduction. Finally, the microorganisms with important characteristics to science will be sequenced to know its identity. While cells plugs were able to be done, the presence of low DNA concentration was present, suggesting incomplete lysis and requiring more optimization of such process.
November 23, 2010
My research projet don’t has result for the moment. I can say that in this moment I am finishing the first metagenomic libraries. It is a group of clone of the Echerichia Coli with the insert (ADN sample) of the soil cell of the Yunque forest. To finish this metagenomic libraries only need the repair reaction, the ligation and the especialized tansduction to indroduce of the ADN sample. Then of make the first metagenomic libraries, I will be of the scanning of the diferent characteristic that I am interested in find. The characteristics that I want scanning are: the biodegradation of the hydrocarbons, the resistance and the production of antibiotic. My research project has like principal proporsal the find and characterize microorganism of the Yunque and the Guanica forest. This characteristics are around of the biomedical and biorremediation field. Then of the realize the first metagenomic libraries, I will begin to make the second metagenomic librarie. This will made like soil sample of the Guanica forest. I want research these diferent forests because I want analyze the microorganisms that be present in these to know their great diversity and diferent function. Some of these fucntions can be important to many aplication. I hope that in this semester I can finish both metagenimic librarie. Also, I hope that I can find some important to public important and interesting resuls.
The Reading of Other Blogs…. October 25, 2010
It is about of synthesize mutations in the acetylcholine receptor that create potential caveolin binding motifs. Also it creates mutations using the Site Directed Mutagensis kit and it is used to measure the function of the receptor after of the mutations are created.
For other part, Lisa Koplick is a sophomore of the University of Puerto Rico, Rio Piedras campus. She is studying a bachelor in Biology. Her research project is based in the biochemistry field with Eric Schreite as mentor. This research project is about of know the structure and function of six α-arrestin proteins. This proteins are the proteins that interacting with Thioredoxin protein (TXNIP). The Thioredoxin is an important protein because this controls the absorption of the glucose in the organism. Already, she works in know the family of the protein of interest, and she has learn all of basic technique.
I think that both students are work in different field but rationed between them. Both research project are very interesting and these shows different new technique that all scientific must learn to can apply in any research.
End of the Semester April 19, 2010
To finish the semester, I can say that in my research project has some conclusion that I want share with you. Some of the conclusions are that in the reaction (1:5:50), the complex of the sulfhemoglobin is formed because you can see a Q band in approximate 620nm. In this specific reaction the absorbance was in a 625nm. This absorbance wasn’t specific 620nm because when the HBI mutant reacts with the hydrogen sulfur and the hydrogen peroxide, it does a series of arrangements inside of the protein causing a variation in the absorbance in the band. Also I can say that this reaction don’t occur satisfactory because in the results you can see a specific bands in 575nm, 540nm, 631nm, and others which represent that in the process of the reaction was presence of the oxygen. This presence of the oxygen broke with the metaquo’s environment desired.
For other part, in this semester I could realize a poster and a presentation about my research project. I am very happy because these experiences help me to development very skills that are necessary in a future. My experience in this semester has been very good because I have learned much knowledge necessary to development as an investigator.
However, I have had some barriers that avoid that I may my research project. Some of these barriers are that I haven’t had much time to make the research. I hope can finish the research project in this semester. I have some plans to continue the research project. Some future goals of the project are the next:
- Make experiments with single HbI mutants and observe the difference or any change.
- Also find how is the velocity of the reactions. This is known as kinetics experiments that make changing the different concentrations of the hemeprotein, hydrogen peroxide or oxygen and hydrogen sulfide.
- Verify the mechanism of sulfhemeglobin complex association and dissociation.
- Experiment using Raman.
- Experiments with double amount of the HbI mutants.
Mid-semester March 6, 2010
An of the technique that I learned durin our research project is inject the solution with a gas. This technique is used to remove the oxygen gas. In our research project, we make the reactions in absence of oxygen. For this reason, we injecte the solutions with a inert gas that remove the oxygen in your sample. We use the nitrogen gas. This is a inert gas where this not react with nothing. We inject all reagents and all materials that have contact with the reaction. For we this technique is very important because we think that the oygen affect much to reaction. I for this that we try remove all oxygen possible.
This research project help me to have a excellent progress. I have achieved at least 50% of the proposed objectives. In our research prooject haven’t had problem to achive my goals. As always, exist moment thar you can’t job in the laboratory. If this occur the work is accumulated and the reseach is
The Beginning of My Second Semester February 5, 2010
I want begin this semester with many force to achieve my objective to shows them all the result. My main objective are:
- Determine factors that control the sormation of the sulfhemoglobin complex using UV-Vis spectroscopy and HbI mutatants.
- Determine the conditions more favorable to a best formation and stability of the sulheme complex.
- Know the mechanism of reaction in sulfhemoglobin derivates formation to help treat the clinical condition know as sulfhemoglobinemia.
We are using the HbI mutants to make rections with hydrogen sulfide and hydrogen peroxide to oobserve the foormation of complex for UV-Vis spectra. I prepared the buffer solution necessary to begin the experiment. Then in next week I will reacts the HbI mutants. After I have the necessary data to present the result in many plece. I will strengthen in this semester are work with the HbI mutants, purify proteins, know and understand the UV-Vis spectra. I like this semester. I can make all the objective to I can show the result in different place.
The end of the semester… November 16, 2009
In this semester I learned many techniques and concept that I helped develop in the field of laboratory. Some of those techniques are:
- Prepare a buffer solution. This is realice with succinic acid. The purpose of this is hold the pH neutral for that the miglobin don’t denatured before or during the reaction for form the sulfohemoglobin.
- Prepare a concentrate solution of mioglobin. Prepare the solution first and later apply pressure with nitrogen for create a solution de few volume but significant concentration.
- Thumb solution and solid with an inert gas like Nitrogen. We need thumb the solution of mioglibin and the solid that give el sulfuric acid to can occur the reaction. The purpose of this is extract the oxygen gas because its reacts with mioglobin and prevent the formation of the sulfohemoglibin complex. First, you create a vacuum and later thumb with nitrogen gas as this is an inert gas which does not form part of the reaction. After of this when realize the reaction obtain a maximum formation of the sulfohemoglobin.
- Also learned use the Apollo system. It is an electronic modern system that determines the exact concentration of a solution. Thus avoid the uncertain data.
During our project we have had several problems with regard to the country status. During this semester had some strikes had that avoid the flow of the project. Notwithstanding all that every time we met and we are working to maximum. For next semester my goals are can continue and find the reality of the sulfohemoglobin. After of finish with the work I want pursue investigating these complex more thoroughly for contribute to the treatment of the sulfohemoglobinemia.
Exploration of a clam and fellowship October 9, 2009
During the last month, I began to work doing a buffer for maintain the ph of the solution in 6.45. This is a neutral pH for that the protein doesn’t denature. Later I am opening the clam to extract the timidia. This is a part of the clam that contain the amino that we need proximally. After of several days I realized a concentrate solution of mioglobin. This solution was realize and after this it is placed in a hub. In those last days were agreeable. My colleagues are very nice. I have a good experience can work with professors with many knowledge that I can learn. I think that the characteristics for occur a good work is the communication, respect, patience and harmony between colleagues. While the most obstacles is can adapt to different personality of the persons. This is form we can realize a successful laboratory.
