Me encantó el contenido de las investigaciones realizadas, los estudiantes dominaban el tema y conocían el propósito de sus invesitgaciones. La experiencia fue extenuante debido a el panel de la mañana y luego el espacio tan estrecho que no te permitía estar en un lugar fijo para escuchar los demás posters (porque habían personas pasando) más los esfuerzos de la voz  para hacerse escuchar.

Ante el requisito de evaluar los posters se observó que estos cumplieron con los requisitos de resumen, introducción, objetivos, metodología y análisis de datos y discusión, con informacion adecuada y letra legible. Entre ellos estan :

Jessian Resto. En su investigación de comparar los habitantes nativos-americanos de la República Dominicana con los de Puerto Rico se encuentran resultados asombrosos. Segun los  de  resultados de Jessian Resto ambos comparten un origen continental común aunque la relacion entre ellos es mínima. Logró excelentes resultados y cumplió con sus objetivos .

Susana Rodriguez con su trabajo titulado Población Microbiana de los lodos biorreactivos en la planta de tratamiento de agua residuales de Mayaguez. Ella dominó tremendamente su tema y me lo explicó muy bien. Su trabajo logró la identificación filogenética de  desconocidos a base del 16s rRna confirmando esta información con las pruebas bioquímicas realizadas. El Segundo objetivo que lograron fue la comparación de individuos identificados por métodos dependiente de cultivos con una comunidad de la misma planta identificado por métodos independientes de cultivo. Se clonó la comunidad seleccionada de la planta de tratamiento de Mayagüez. Se  mejoró las técnicas moleculares en el laboratorio aparte que mejoró las  habilidades de “networking” con el profesor y el estudiante graduado en su laboratorio.

Y finalmente Manuel Ortega con su trabajo titulado Screening and Isolation of novel biological active molecules with possible biotechnology applications from Metagenomic Libraries of different Forest’s soils in Puerto Rico. Hace un screening y una separación de nuevas moleculas biologicas con posibles aplicaciones al a biotectnología desde Bibliotegas de diferentes suelos de bosques en Puerto Rico hasta la fecha 5,850 clones de el Yunque se han leido y no han encontrado actividad anti-hongos bajo las condiciones experimentales que se han trabajando. Con la separación de un clon positivo de la biblioteca en su trabajo se procedió a caracterizar su gen e identificar si el compenente anti-hongo también inhibe el crecimiento de otros hongos patogénicos. Su trabajo me pareció algo interesante.

We still need to do analyses and work with some stuff. Sometimes I feel lost in the work and don’t understand the slow advaces we do. We had problems with the materials, we haven’t all of them and it retarded the work. To deal with the problem of the materials we ran all the University looking for some reactives that we need in the Biology or Chemistry departments.

Abstract

Generation of GFP Variants using Error Prone PCR and

Characterization in Escherichia coli

Genetic variants can be generated through gene modification. The main interest of this research is to generate new variants of the Green Fluorescent Protein (GFP) with novel properties. GFP is a protein, comprised of 238 amino acids (26.9 kDa), found in the jellyfish Aequorea victoria that fluoresces green when exposed to blue light. Escherichia coli is frequently used in Biotechnology because it is relatively easy to work with and its genome has been fully characterized. We used an Error-Prone PCR technique predicted to be effective at generating grossly divergent sequences. PCR is carried out under specific conditions with increased concentrations of manganese allowing the Taq polymerase to mismatch incorporated nucleotides during DNA replication. GFP is useful as a marker to study when proteins are made and where they go. Fusion genes are created by joining the GFP gene to the gene of the protein of interest so that when the product is made it will encode a fusion protein. Our goal is to generate, using error prone PCR, variants of GFP with novel properties that could be more resistant to extreme environments or show variations in fluorescence properties. We first used bioinformatics to analyze the pGLO plasmid sequence containing GFP and designed PCR primers to amplify the gene encoding this fluorescent protein. Using these primers, we amplified GFP using normal (N) and error prone mutagenic (M1 and M2) PCR conditions. PCR amplification of pGLO DNA under both normal and mutagenic conditions produced amplified products of the predicted size that were detected using agarose gel electrophoresis and purified for cloning. We ligated these purified normal and mutagenic GFP inserts into the cloning vectors pGEM-T Easy (Promega) and pETBlue-1 (Novagen). Subsequently we transformed the ligation products into E. coli JM109 and analyzed the white colonies containing the GFP inserts by preparing plasmid DNA, digesting with appropriate restriction endonucleases, and analyzing the resulting DNA fragments using agarose gel electrophoresis. DNA sequences were determined for four fluorescent GFP pGEM-T Easy clones and some are currently being analyzed for amino acid changes. We are also studying the fluorescent properties of these GFP pGEM-T Easy clones. A rapid screening of randomly generated mutant fluorescent GFP proteins using the pET-Blue1 vector is currently being developed. We thank the UPR-Cayey Biology Department, RISE Program (NIH GM 59429), and the BioMinds Program (funded by the Amgen Foundation) for supporting this research.

The goals for this semester are :

1. Screen random mutant GFP molecules to identify those with novel properties.

2. Analyze the sequences of the GFP genes cloned using normal and mutagenic PCR conditions.

3.  Characterize the GFP Flourescence in Escherichia coli.

The work of this semester will be analyzing the results of the last semester. We already generated new variants of GFP in Escherichia coli.

For this semester we are going to work over some techniques:

Transformation
Purification
Ligation
Characterization of the fluorescence of GFP (Not sure of the technique yet)

About the new techniques I’m not sure with precision we are going to do. Today we meet and during this week we are going to be talking with our mentor about it.

·

The Significance of my work is :

• GFP is useful for label.
• Target because since GFP fluoresces one can shine light at the cell and wait for the distinctive green fluorescence associated with GFP to appear.
• Using error prone PCR we are creating new variants of the GFP.
• E. Coli is useful because they are relatively easy to work with and because there is a vast amount of previous knowledge about them.

Talking about the Specific Aims they are :

I. New Up Primer design at ATG initiation codon of the GFP gene in pGLO.
II. Amplify the GFP region under normal and mutagenic conditions.
III. Clone the GFP amplified region into the pET-Blue1 Vector.
IV. Analyze GFP variant protein properties.
V. Characterize GFP variants in E. coli.

Using the Specific Aims established for this semester. I can say that the progress we have done can be classified in number 2. That means  we have done lees of a 50% of our main Goals. The specific aims that we have accomplished are : the I .New Up Primer design at ATG initiation codon of the GFP gene in pGLO, and
II. Amplify the GFP region under normal and mutagenic conditions.

This picture taken in October 23rd 2008  shows the results of a PCR under normal and mutagenic conditions

Lane 1 – ladder 100bp
Lane2 – old primer used
Lane 3- New primer at ATG initiation codon of the GFP gene in pGLO.
Lane 4 – Mutagenic PCR round 1
Lane 5 – Mutagenic PCR round 2
Lane 6 – Ladder 1 kbp

• Prepare a Polymerase Chain Reaction GFP.
• We are going to be analyzing and characterizing the GFP protein and its variants generated last semester.
• We are going to use a shuttle vector for the expression of GFP

This work is in sequence of the last semester. We are repeating the PCR because decoding the mutant of GFP we had difficulties. We also had results that are mutants of GFP that are going to be characterizes during this semester.

I have learned new techniques in the laboratory. I learned about how to improve some techniques that I already knew. I have learned that the research requires a lot of dedication.

II. What barriers and challenges you have to confront for began to realize you research?

The economy was a challenge because we have to work and deal with the materials that we have. For example is will be greater a gel of acrylamide than an agarose gel but the budget and the equipment don’t allow us that. Also a barrier has been the place where we sent our DNA for receive the sequences. Because we still waiting, and probably we have to make again mini preps because of the University that don’t sent us all the sequences of the DNA that we sent them.

III. How have you overcame the barriers and challenges?

With patience and acceptation of the reality of the research.

IV. What are do you expect for next semester?

I expect to have the sequence of the mutagenic DNA of GFP for made a comparison between the original and the mutagenic that we created. Also I expect to begin the characterization of the protein GFP.

PCR

Fig 1 ; PCR Machine

The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.

Ligation

Fig 2 DNA ligase with DNA

DNA ligases have become an indispensable tool in modern molecular biology research for generating recombinant DNA sequences. For example, DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids.

One vital, and often tricky, aspect to performing successful recombination experiments involving ligase is controlling the optimal temperature. Most experiments use T4 DNA Ligase (isolated from bacteriophage T4) which is most active at 25°C. However in order to perform successful ligations, the optimal enzyme temperature needs to be balanced with the melting temperature T_m (also the annealing temperature) of the DNA fragments being ligated.

If the ambient temperature exceeds T_m, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding. The shorter the DNA fragments, the lower the T_m. Thus for sticky ends (overlaps) less than ten base pairs long, ligation experiments are performed at very low temperatures (~4-8°C) for a long period of time (often overnight).

The common commercially available DNA ligases were originally discovered in bacteriophage T4, E. coli or other bacteria.

Transformation

Is the genetic alteration of a cell resulting from the uptake and expression of foreign genetic material (DNA). Separate terms are used for genetic alterations resulting from introduction of DNA by viruses (“transduction”) or by cell-cell contact between bacteria (“conjugation”). Transformation of animal cells is usually called transfection.

The term transformation is also used more generally to describe mechanisms of DNA and RNA transfer in molecular biology (i.e. not only the genetic consequences). For example the production of transgenic plants such as transgenic maize requires the insertion of new genetic information into the maize genome using an appropriate mechanism for DNA transfer; the process is commonly referred to as transformation.

Most part of the information posted in this blog have been taken from Wikipedia.org

We began last semester generating the new variants of GFP and during this semester we are doing to be studying and analyzing different variants of the generated last semester.

I work with the same mentor and share the information about our own researches with Angiemar Maldonado. She is a friend and we take courses together. I receive recommendations of her about my research and she know I’m available for help her. I think is very important the responsibility and commitment in the research; is easier to work with people who have similar visions of life. I’m not having difficult working as a team with my Biominds group and I really like the people that I’m working with.