Visiting other biopeers’ sites allowed me to get to know their research projects and know the diversity of projects available at my campus. I was able to ask questions to other peers about their research and discuss differents aspects of their projects.
Insights into the world of molecular biology… March 25, 2008
Fap1 is the yeast homologue of the human protein NF-X1 that competes with the drug rapamycin and confers resistance to it. In yeast, rapamycin resistance is achieved when Fap1 binds the protein FKBP12 that allows the continuation of the mTOR protein pathway that induces cell cycle progression in the presence of nutrients. One important characteristic of Fap1 that is shared with its human homologue is the presence of zinc-finger domains. Zinc-fingers are formed with the presence of a cystein-rich region in the amino acid sequence of the protein that is able to coordinate with zinc ions to form a finger-like structure that can bind DNA. This property allows the protein to bind to a DNA molecule suggesting the possibility of acting as a transcription factor like the human homologue. The objectives of the project are to clone the FAP1 gene into a vector for later expression in bacteria to perform crystallographic studies of the zinc-finger regions of the protein. Using the zymolase reaction technique, we were able to obtain the PCR fragment of the gene. The zymolase reaction exposes the genetic material from the S. cerevisiae nucleus and permits the amplification of the gene. The gene was successfully cloned into the pGEM-T Easy Vector which was characterized by the method of restriction enzyme (RE) digestion analysis. To facilitate the process of crystallization, truncated versions of the protein are to be made by cutting a section of the DNA sequence and substituting that section with a 50 bp-long fragment that would shorten the FAP1 sequence by 1300 bp. The RE used were MfeI that cuts at the last zinc-finger region and AflII that cuts at the vector region after the FAP1 gene terminator codon. To the new fragment that will be inserted, a terminator codon sequence was inserted to stop translation of the gene, thus obtaining a truncated version of the protein. Currently, the pGEM-FAP1 construct has been digested with the corresponding RE to obtain a linear truncated DNA fragment. The digestion was run on a 1% agarose gel electrophoresis, the larger fragment was extracted and purified with a gel purification kit. Now, we are testing ligation reaction efficiency to perform the ligation reaction of the truncated construct and the synthesized oligonucleotide fragments.
