Library Generation and Auxotrophic Selection Assays in Escherichia coli and Thermus thermophilus

The selection of optimized enzymes from gene libraries is important, both for basic and applied research. Here, we first describe the generation of plasmid-borne libraries using error-prone PCR and highly competent Escherichia coli cells. We then provide protocols for the use of these libraries for auxotrophic selection assays with E. coli and the extremely thermophilic bacterium Thermus thermophilus as hosts.