Directed in vitro evolution of reporter genes based on semi-rational design and high-throughput screening.

Marker genes, such as gusA, lacZ, and gfp, have been applied comprehensively in biological studies. Directed in vitro evolution provides a powerful tool for modifying genes and for studying gene structure, expression, and function. Here, we describe a strategy for directed in vitro evolution of reporter genes based on semi-rational design and high-throughput screening. The protocol involves two processes of DNA shuffling and screening. The first DNA shuffling and screening process involves eight steps: (1) amplifying the target gene by PCR, (2) cutting the product into random fragments with DNase I, (3) purification of 50-100 bp fragments, (4) reassembly of the fragments in a primerless PCR, (5) amplification of the reassembled product by primer PCR, (6) cloning into expression vector, (7) transformation of E. coli by electroporation, and (8) screening the target mutants using a nitrocellulose filter. The second DNA shuffling and screening process also involves the same eight steps, except that degenerate oligonucleotide primers are based on the sequence of the selected mutant.