Team:Johns Hopkins/Project

As our inaugural project, we set out to simplify one of the somewhat belaboring tasks in the field of molecular biology: S. cerevisiae mating type elucidation. Baker’s yeast, S. cerevisiae, has become an invaluable eukaryotic model for molecular biology research for many reasons. One of the main reasons is its utilization of proteins homologous to those found within humans, as well as many of the same biochemical pathways. Investigating the biochemical systems in yeast has provided insight on various genetic diseases found in humans. Also, because they are unicellular and therefore grow fast, they can be studied more readily than many other cell lines, especially those of higher eukaryotes. Another useful characteristic of yeast is its ability to exist in populations of different ploidy, either diploid or haploid. The process in which ploidy arises is governed by the yeast mating pathway, and is well studied. A haploid yeast cell is either mating type ‘a’ (MATa) or mating type ‘α’(MAT α). In the elucidation of biochemical and genetic processes in yeast, many times it is crucial to initiate sporulation of diploid yeast cells. After sporulation occurs, there are four haploid cells; two MATa and two MAT α. To continue analysis, usually differentiation between these cells is crucial, and this process can take from 2 to 3 days. We propose to cut this time by creating a plasmid containing fluorescent proteins that would allow for visual determination of S. cerevisiae mating type. To do so we would utilize the existing regulator proteins control of the expression of specific fluorescent proteins

Depending on the specific regulators being produced, a cell will exhibit a given sex, either MATa or MAT α. (Herskowitz 750) The MATa associated products are controlled by, α1 and α2 regulators. The α1 regulator promotes the transcription of MAT α associated products and the α2 regulator inhibits the production of MATa associated factors. In absence of MAT α regulators, the MATa cells produce only MATa associated factors, without MAT α associated factors due to a lack of inhibition by α2, and a lack of α1 to initiate the production of MAT α associated factors. (Sprague 959) The a1 regulator that is produce by MATa cells is only utilized when in combination with a2 to inhibit specific regions, associated with a MATa/ α diploids. The design of the construct would be such that a MAT α cell would produce only one fluorescent protein, and in the case of the prototype diagram of our construct, green fluorescent protein (GFP). A MATa cell would produce both fluorescent proteins, GFP and red fluorescent protein (RFP), thus yielding a different color. Also a diploid cell, would be detectable as well due to lack of color, due to regions before each fluorescent protein open reading frame that are inhibited by the diploid a1-α2 combination factor. In addition to these control regions, before both fluorescent protein open reading frames there would also be a promoter region, foreign to yeast, most likely from bacteria. This would allow the investigator to be in control in the production of these sex-dependent fluorescent proteins, such that if protein fluorescence is used to study another aspect of yeast physiology, the sex dependent florescence would be inactivated. By utilizing the specificity in genome control between the sexes of the haploid yeast cells, along with a specific region of control in diploid cells, the creation of a reliable, novel, and practical detector would be achievable.