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¡Hola BioCompañeros!

 

Muchos saludos de mi parte. A continuación les coloco mi abstract.

 

Saccharomyces cerevisiae expresses FAP1 protein during cell cycling progress.  This protein has a human homologue called NF-X1, identified as a transcription factor involved in the regulation of MHC-Class II genes. NF-X1 limit inflammatory responses by negatively regulating the transcription of these genes. FAP1 is localized in the cytoplasm but, upon rapamycin treatment, translocates to the nucleus. These findings therefore suggest a nuclear role for this novel FKBP12 ligand, possibly as a transcription factor. Also, based on their amino acid sequence, both proteins contain a DNA-binding domain called the zinc finger.  This domain is required for many eukaryotic proteins in order to bind DNA for the expression of certain genes.   In this research project, a FAP1:pGEM construct was used for transformation of electrocompetent BL-21 cells and plasmid Bluescript as a control.  Transformants were first grown in LB media containing ampicillin at 37°C until reach an OD₆₀₀ of 0.5.  IPTG was later added for a final concentration of 100µg/mL to induce the expression of corresponding proteins for 4 hours.  The bacterial culture was harvested before the preparation of crude extracts. After the crude extracts were prepared, the total protein concentration was determinate by a BCA protein assay and read using a spectrophotometer.  The expression of FAP1 and b-galactosidase was monitored with SDS-PAGE. The proteins bans were visualized by Silver Staining and Coomasie Blue   However, since both proteins co-migrate on the same gel and the observed protein bands were very weak, we are now working on a protocol for the preparation of stable protein extracts.  This presentation will include the new methodology proposed by (S. Otnan. 1996) with minor modifications. This work was supported by Bio-MINDS Program sponsored by The Amgen Foundation.

 

Desde el mes pasado, el progreso que he conseguido en base a mis metas es un 4 (mucho progreso, he logrado al menos 80% de los objetivos). Hemos avanzado bastante. Lo único que me falta es esperar unos materiales y proseguir con mis experimentos a ver si puedo lograr el objetivo principal de mi trabajo (expresar a FAP1). Básicamente las dificultades han sido esperar mis materiales. Mientras tanto sigo leyendo más literatura y haciendo unos ensayos para usarlos como control. Estoy transformando Bl-21 Cells con pBKS para expresar Beta-galactosidasa y ver si este método me ayudará con mi proteína. Sigo motivado y confiado en nuestro trabajo. Espero que todos tengan mucho éxito y los veré en la presentación. Cuídense…