Presentation description

Acetyl-CoA (AcCoA) is found naturally throughout the cell as a core metabolite and beyond. Existing methods of AcCoA detection are limited in use due to their reliance on lysates, which eliminate the possibility of live cell-tracking. A fluorescent biosensor would offer a better alternative, since it would have higher throughput and be suitable for live cell imaging. Constructed from PanZ and cpGFP, PancACe 1.0 has been successful in detecting AcCoA in HeLa cells and in titrations. With a KD of 274 µM, its dynamic range corresponds more closely with E. Coli than mammalian cells, which range from 1-50 µM. Modifications have since been made to the sensor to improve quantifiability and sensitivity in mammalian cell trials via an mCherry addition and a binding site mutation, referred to as PancACe 2.0. Binding affinity for AcCoA increased 11-fold to 25 µM, with a dynamic range of 0.1-100, but acyl-CoA specificity was lost as PancACe 2.0 affinity for propionyl-CoA became nearly identical to that of AcCoA. From looking at protein models, acyl-CoA specificity may be increased by restricting the back of the PanZ binding pocket. To achieve this, we made point mutants which fill that space.