Optimization of protein-based volumetric optical memories and associative processors by using directed evolution

The potential use of proteins in device applications has advanced in large part due to significant advances in the methods and procedures of protein engineering, most notably, directed evolution. Directed evolution has been used to tailor a broad range of enzymatic proteins for pharmaceutical and industrial applications. Thermal stability, chemical stability, and substrate specificity are among the most common phenotypes targeted for optimization. However, in vivo screening systems for photoactive proteins have been slow in development. A high-throughput screening system for the photokinetic optimization of photoactive proteins would promote the development of protein-based field-effect transistors, artificial retinas, spatial light modulators, photovoltaic fuel cells, three-dimensional volumetric memories, and optical holographic processors. This investigation seeks to optimize the photoactive protein bacteriorhodopsin (BR) for volumetric optical and holographic memories. Semi-random mutagenesis and in vitro screening were used to create and analyze nearly 800 mutants spanning the entire length of the bacterio-opsin (bop) gene. To fully realize the potential of BR in optoelectronic environments, future investigations will utilize global mutagenesis and in vivo screening systems. The architecture for a potential in vivo screening system is explored in this study. We demonstrate the ability to measure the formation and decay of the red-shifted O-state within in vivo colonies of Halobacterium salinarum, and discuss the implications of this screening method to directed evolution.