Preparing Libraries in Escherichia coli

The process of preparing libraries of mutagenized or recombined gene sequences for screening or selection in Escherichia coli is a special application of cohesive-end subcloning (1 ). PCR products are digested with restriction endonucleases, ligated into an expression vector digested with the same enzymes, and the resultant recombinant plasmids are transformed into supercompetent bacteria. One difference between routine subcloning of a single gene and preparing libraries is that in the latter case, there can be little allowance for the presence of transformants containing recircularized vector and no insert (so-called “background” ligation products). Furthermore, ligation and transformation of the recombinant plasmids must be performed using materials and conditions that yield a sufficient number of transformants (∼10³ –10⁵ ) for identifying variants exhibiting desired properties.