 Hi I'm Colin Jackson and this is Jason Woodfield. This is a video abstract for our paper construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction. Jason will now take you through some of the technical details and interesting bits of the paper. The construction of genetically encoded biosensors generally relies on the availability of a binding core. In this case a member of the paraplasmic binding protein family that is specific for the target ligand and also stable such that measurements can be made in physiologically relevant conditions. This binding core can then be combined with a pair of fluorescent molecules to create a flip sensor. This class of proteins is an attractive option for sensor design owned to their significant confirmation change upon interaction with the ligand. The flip sensors work via a forced-resonance energy transfer mechanism or FRET such that the efficiency of transfer of energy from the donor, Shona Green, to the acceptor Shona Red is dictated largely by the interfluorophore distance. L-arginine was the target molecule for our study and as the immediate precursor of nitric oxide it is an important molecule in many physiological contexts including vasodilation and wider brain function. Binding proteins with the desired properties are not often available in nature and substantial improvement sensors can be acquired particularly with regard to the durability. So in our work we use a combination of ancestral protein reconstruction and circular permutation to create a suitable biosensor. Ancestral protein reconstruction is a powerful protein engineering tool that would generate highly stable and functional proteins by the construction of a hypothetical phylogeny for a given protein family. Due to the increased stability the ancestral binding core is robust to further engineering such as circular permutation. Technique best visualized as a linear protein sequence where the N and C terminal are fused by a flexible linker and the sequence is cleaved in a new location thus creating new terminal regions. This technique has been used before by Carter et al in their 2009 paper where they applied it to other PPPs to improve the dynamic range of flip sensors by creating new terminal. Outsights with a conformation change of the PPP has the greatest potential to cause a change in FRET. In this case the hinge region as illustrated by panel B. Our sensor C-P flipper displays high sensitivity and specificity with a KD of approximately 14 micromolar and a maximum dynamic range of 35%. Importantly C-P flipper was highly robust enabling allergenine measurement at physiological temperatures and intact brain tissue using multi-photon excitation. Our in situ measurement of allergen showed a concentration of approximately 17 micromolar which is close to previously published data of 18. Thank you for watching our video we hope that the sensor will be useful for others in the field and be used to better understand the role of arginine and signaling and disease.