I think we are at #3 = a 50% of progress about the objectives presented at the begging of this semester.

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.