Abstract
Publisher Summary This chapter discusses the use of directed evolution of bacteriorhodopsin (BR) for device applications. This protein is synthesized by the salt marsh archaeon Halobacterium salinarum when oxygen availability drops below a level sufficient to sustain respiration. The organism then switches spontaneously to photosynthetic energy production by generating a purple membrane containing BR. It is found that when BR absorbs light it pumps a proton, and the pH gradient is utilized to convert ADP to ATP. Site-directed mutants are constructed using the Stratagene Quik-Change mutagenesis system. Construction of BR variants for expression in T. thermophilus is achieved using a combination of mutagenesis methods. Inserting mutant bop fragments into the multiple cloning site of pMKE1 is the final step in the construction of a BR thermoselection vector. A combination of mutagenesis techniques are used to create randomly mutated regions in the bop gene. It is found that the thermal stability of BR is achieved by expressing randomly mutated proteins in an extremely thermophilic background.
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