~*~*~ Progreso y Abstract, Febrero 2009~*~*~

Evaluando esta experiencia en lo que va de semestre entiendo que he alcanzado un 3 en mi nivel de progreso. Hasta el momento he logrado realizar diluciones de una biblioteca ya creada por el laboratorio y realicé pruebas de proteasa y quitinasa en un “subpool”de la biblioteca Rainy Bottom. En este momento me dispongo a repetir estas pruebas ya que obtuvimos resultados negativos. Por otra parte, continúo trabajando en la generación de bibliotecas de bajo peso. Ya realicé 4 extracciones para determinar la muestra con mayor cantidad de DNA para continuar usándola este semestre.Cabe señalar que he tenido algunas dificultades en este semestre ya que me encuentro en mi último semestre y he estado viajando a entrevistas a distintas universidades dentro y fuera de los Estados Unidos ya que solicité a programas de PhD. en Ciencias Biomédicas. Por esta razón no he tenido el tiempo suficiente para asistir con regularidad al laboratorio. Además, para realizar otros monitoreos planificados necesito algunos reactivos y “primers” que estoy esperando a que sean entregados durante esta semana.

Ya terminé mis entrevistas, por lo que espero poder reanudar mis experimentos y labores en el laboratorio y adelantar mis experimentos atrasados. Además, en cuanto lleguen mis reactivos voy a trabajar extra para poder tener algunos resultados interesantes para este semestre y para presentar en el afiche de BioMinds.

Adjunto presento mi resumen del afiche que presentare en el BioMinds Poster Day!

Generation of Small Size Metagenomic Libraries from a Tropical Rain Forest in Puerto Rico and Functional Screening of the libraries using enzyme activity and genetic Engineering Tools

R.K. Rivera, Z.M. Corales and C. Rios. University of Puerto Rico-Mayagüez. Biology Department.

Metagenomics is an emergent field in which genomic analysis is applied to entire microbial communities surpassing the need of isolation and cultivation of microbial communities or individuals, and allowing researchers to study genomes in a community as a whole. While traditional methods are restricted to cultivable organisms, metagenomics allows the study of microorganisms that account for 1% of the microbiota of our planet and that do not grow under laboratory conditions. This project focuses on: 1) the generation of small size metagenomic libraries using the microenvironment of a soil sample from Pico del Yunque, a forest in Puerto Rico, and 2) screening of these libraries for different enzymatic capabilities. Using a direct extraction approach, genomic DNA was isolated from the forest soil samples and after digestion of this DNA with endonucleases, fragments of 4kb – 10kb were excised, purified, cloned into pCF430 vectors and transformed into Escherichia coli Epi300 strains. The transformed cells were plated on Petri plates with media containing tetracycline. Several clones were isolated and current experimental approaches have been done in order to extract and digest the recombinant plasmid in order to detect the presence of a cloned fragment, to further perform an in silico analysis. There are also efforts to optimize the methods in order to increase the number of clones with the desired fragment. The second part of this project involves the screening of large size insert of metagenomic libraries previously generated in the laboratory. Subpools of a tropical forest metagenomic libraries were screened for the protease activity resulting in possible clones that are currently being tested to verify their identity. Further screening procedures will be performed to search for DNAse activity as well as sequence analysis for the presence of other enzyme sequences. Specifically, and using PCR we will search the libraries molecularly to detect the presence of genes which encodes for the activities of enzymes such as α-amylase, 4-hydroxybutyrate dehydrogenase and poly(3-hydroxyalkanoate) synthase. The importance of these libraries lies in that they will help unravel important enzymatic activities with biomedical and biotechnological applications from a very distinctive forest while technology with natural resources conservation are reconcile.

Funded by Amgen® Biominds at UPR and MARC/Sloan Programs at UPRM.

Category: Biotechnology

Deja un comentario