[16] Methods for mutagenesis of the bacterioopsin gene

This chapter discusses methods for mutagenesis of the bacterioospin gene. Most of the bacteriorhodopsin structure is of undetermined function, procedures involving generalized mutagenesis are potentially useful in the initial stages. To provide an easily selectable phenotype, a fusion between the genes for bacterioopsin and β-galactosidase is constructed on a multicopy plasmid, pXB/Gal 101. The fusion gene, containing the bacterioopsin gene (bop) fused upstream from the β-galactosidase gene, is under the control of tandem lipoprotein and lac gene promoters. Deletion mutagenesis has been widely used to define functional domains in DNA and proteins. In bacteriorhodopsin and other polytopic membrane proteins, this approach could be used to test structural models and to investigate systematically the size of the loops protruding into the aqueous medium and of the domains embedded in the bilayer. Point mutagenesis can be achieved by a number of procedures, which involves cloning the gene of interest into M13 and using single-stranded DNA from this clone as template for in vitro primer extension with the mutagenic oligonucleotide as primer. This yields double-stranded DNA which should segregate to give 50% mutant clones.