This summer the interns worked on a project to find mutations in the scaffold protein Sterile5 that altered the quantitative system output of the alpha mating pheromone response pathway. To make the mutations the interns used error-prone PCR to introduce random mutations in the Ste5 gene. They used specially designed PCR primers to amplify DNA encoding specific functional regions of the Ste5 protein (e.g. plasma membrane binding domain, Fus3 binding domain, Ste7 binding domain and Ste11 binding domain) and introduced the mutated DNA segments back into a plasmid containing an otherwise normal Ste5 gene by homologous recombination. They transformed the plasmids into a yeast strain in which the native Ste5 gene was deleted. The strain also contained a reporter gene fusion that conferred histidine auxotrophy in response to pheromone. To eliminate mutations that rendered the Ste5 protein non-functional, the interns first selected for transformants that grew on plates with alpha pheromone but without added histidine. The assumption was that strains with a functional alpha mating response pathway (and therefore a functional Ste5) would be able to grow on this selective media. They then screened for mutants with altered responses to alpha pheromone (e.g. increased or decreased sensitivity) by monitoring the growth rate of single colonies on plates with alpha pheromone and without histidine (along with a few other key chemicals that are described in their presentation).The interns found many candidates with both an increased and decreased rate of growth and followed up with a few of the most interesting ones

The interns worked really hard throughout the summer and learned many new techniques including PCR, gap repair cloning, replica plating, 'frogging', halo assays, and fluorescence microscopy.They did a fantastic job all summer long. At the end of the summer the interns presented their work to their parents, friends and MSI scientists. To learn more about 2007 internship project download and view their presentation (Download PowerPoint slides -11MB file )