En mi investigación durante este semestre he afrontado varios problemas por eso le doy un 2. Entre esos problemas está el que empezamos tarde el semestre y ya pronto es el día de presentar los afiches. Esto crea problemas porque tenemos que hacer dos cosas a la misma vez y se dificultad volver a repetir los experimentos porque tenemos una agenda con experimetos bastante cargada. Para resolver esto hemos planeado los experimentos muy bien y de todos modos hay factores que son dificiles de controlar por lo hay casos en los que si tenemos que repitir y se atrasan los demas experimentos. Otro factor es muchas veces hay mucho conflicto con las clases y los profesores, porque deciden dar reposiciones de clases a las que faltan en horas que usualmente se utilizan para hacer experimentos.

Screening and Analyses of Error Prone PCR Generated
Green Fluorescent Protein (GFP) Variants
Introduction: Biological engineering is a form of synthetic life, since specific DNA components are introduced into an organism’s genome.  The modified organism will respond as it is programmed.  The Green Fluorescent Protein (GFP) produces light by fluorescence that is emitted from its fluorophore center composed of the Ser-65, Tyr-66, and Gly-67 amino acid sequence.  Methods: The pGLO plasmid containing GFP was purified using the Alkaline Lysis Plasmid Purification method, the GFP gene was verified by restriction digestion of pGLO plasmid using the restriction endonucleases BamHI and HindIII, and the digested plasmid visualized using agarose gel electrophoresis.  We used Bioinformatics techniques and sequence databases to design primer sequences, using the Primer3 Internet program, that were used to amplify the GFP gene from the pGLO plasmid using the polymerase chain reaction (PCR).  We performed PCR under normal conditions (N) and multiple cycles of Error Prone PCR to create GFP mutagenized variants.  In the Error Prone PCR, the PCR conditions are altered in order to cause random errors when the Taq polymerase replicates the template strand.  The first cycle of mutagenic PCR (M1) was amplified directly from the pGLO plasmid template.  In the second cycle of mutagenic PCR (M2), the product from the first mutagenic cycle was mutated again under error prone conditions.  Then the amplified PCR fragments were purified and the results analyzed using agarose gel electrophoresis. We then ligated the purified PCR fragments to the cloning vectors pGEM-T Easy (Promega) and pET-Blue1 (Novagen).  The vectors ligated to GFP were transformed into E. coli JM109 competent cells to screen for fluorescent colonies that contained the insert (white colonies).  We purified plasmid DNA from GFP clones, used enzymatic digestions to verify the cloning, and the DNA sequence of selected clones were determined.  Results: The pGLO plasmid DNA was purified and three DNA fragments were obtained after restriction digestion with the enzyme BamHI, and two with HindIII confirming its identity as the pGLO plasmid.  Specific GFP upstream and downstream primers were designed based on the downloaded pGLO gene sequence encoding the GFP protein.  PCR reactions using various sets of designed primers amplified GFP fragments of the expected size.  These PCR amplified GFP DNA fragments were successfully cloned into pGEM-T Easy and pET-Blue1 vectors for analysis as shown by agarose gel electrophoresis of mini prep plasmid DNA.  DNA sequence analysis of one of four fluorescent GFP pGEM-T Easy clones revealed a double mutation resulting in two amino acid substitutions (Asn171Asp, Tyr238Asn) whose fluorescent properties are currently being determined.  Conclusions: A rapid screening of randomly generated mutant fluorescent GFP proteins using the pET-Blue1 vector is currently being developed.  In the future, the variant GFP DNA will be inserted in Halobacterium using the shuttle vector pNG168 in order to characterize the properties of the mutated GFP proteins in extremophiles.  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.