Presentation description

Acetyl-coenzyme A (AcCoA) is a vital molecule in mammalian cells. It is commonly known for connecting glycolysis to the TCA cycle but is also heavily involved in gene regulation and the synthesis of fatty acids and cholesterol. Current methods to measure AcCoA levels include the PicoProbe - an enzyme coupled assay, and various mass spectrometry-based assays. However, these methods present limitations, such as being unsuitable for live cell imaging due to reliance on cell lysates. Recently, a genetically encodable biosensor, PancACe, was developed by combining the binding protein PanZ with fluorescent protein cpGFP. This biosensor addressed limitations from previous methods, but had some shortcomings of its own, mainly low affinity and difficulty in quantification. To address these shortcomings, mutations were introduced to created PancACe 2.0, which has a more suitable dynamic range for mammalian cells and fluorescent quantification to normalize measurements. Our research focuses on using PCR-based site-directed mutagenesis to modify PancACe 2.0 and improve the biosensor's affinity for acetyl-CoA, making it more selective against the structurally similar propionyl-CoA.