PROJECT SUMMARY/ABSTRACT We propose to develop fully autonomous “self-driving laboratories” to rapidly engineer enzymes for broad applications in biomedicine and biocatalysis. Our approach mimics the methodology of a protein engineering researcher with an AI layer that builds an understanding of protein sequence-structure-function and plans experiments to test specific protein design hypotheses, and a robotic system that experimentally tests designed proteins by synthesizing genes, expressing proteins, and performing biochemical measurements of enzyme activity. Seamless integration between the intelligent agent and experimental automation enables fully autonomous design-test-learn cycles to understand and optimize the sequence-function landscape. Self- driving laboratories will revolutionize the fields of biomolecular engineering and synthetic biology by automating highly inefficient, time consuming, and laborious protein engineering campaigns, enabling rapid turnaround, and allowing researchers to focus efforts on important downstream applications.