Cellular energy is a cornerstone of metabolism and is crucial for human health and disease. Knowledge of the cellular energy states and the underlying regulatory mechanisms is therefore key to understanding cell physiology and to design therapeutic interventions. Cellular energy states are characterised by concentration ratios of adenylates, in particular ATP:ADP and ATP:AMP. We applied synthetic biology approaches to design, engineer and validate a genetically encoded nano-sensor for cellular energy state, AMPfret. It employs the naturally evolved energy sensing of eukaryotic cells provided by the AMP-activated protein kinase (AMPK). Our synthetic nano-sensor relies on fluorescence resonance energy transfer (FRET) to detect changes in ATP:ADP and ATP:AMP ratios both in vitro and in cells in vivo. Construction and iterative optimisation relied on ACEMBL, a parallelised DNA assembly and construct screening technology we developed, facilitated by a method we termed tandem recombineering (TR). Our approach allowed rapid testing of numerous permutations of the AMPfret sensor to identify the most sensitive construct, which we characterised and validated both in the test tube and within cells.
Keywords: ACEMBL; ADP; AMP; AMPK; ATP; biosensor; cellular energetics; fluorescence resonance energy transfer.
© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.