Quantitative approaches to DNA-protein interactions using synthetic biology, high-throughput yeast one-hybrid assays and structural modelling of protein and DNA

Professor David FitzPatrick

Mutations in the PAX6 transcription factor result in human eye malformations.  We will use synthetic biology technologies to construct a library of plasmids with all possible amino acid substitutions in the disease-associated paired domain.  We will employ yeast one-hybrid (Y1H) technology to assay the binding ~2600 variants of the PAX6 paired domain to three validated vertebrate PAX6 binding sites .  This will generate a quantitative measurement that correlates with the DNA binding potential of each variant. This high-throughput approach will allow us to better understand how PAX6 mutations cause disease at a molecular level, and predict new disease-causing mutations.