"In Vivo" Platform Plasmid Method using "Saccharomyces cerevisiae" for Determining Binding Affinity Profiles of Transcription Factors
- Author(s): Cugini, Alexis Julia
- Advisor(s): Rifkin, Scott
- et al.
While transcription factors are a crucial part of gene regulatory networks, their role in evolutionary processes is still not fully understood by researchers. Recent advancements in studying protein-DNA interactions have revealed that transcription factors have a binding affinity profile where there are both primary and secondary binding sites. The existence of these binding profiles hints to the possibility that transcription factors may have more potential for change than previously thought. How binding profiles may be affected by changing environmental conditions or mutations in the binding domains and binding sites remains to be seen. Existing in vitro and in vivo methodologies do not have the ability to produce the high throughput, informative data required to answer these questions. In this paper, an inducible platform, integrated in the yeast genome, is described that allows for the study of multiple transcription factors in vivo. By measuring an observable phenotype (fluorescence), how introduced mutations and altered environmental conditions affect the binding abilities of a chosen transcription factor can be investigated. The platform plasmid has already been assembled and the inducible system has been transformed into the yeast cells. The next steps are to transform the platform plasmid into the yeast and to insert a target transcription factor system for a pilot study. Once completed, this system will allow for more comprehensive studies of the binding space of various transcription factors.