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.
Xiong
Ai-Sheng
AS
Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, China. xiongaisheng@saas.sh.cn
Yao
Quan-Hong
QH
Peng
Ri-He
RH
Cheng
Zong-Ming
ZM
D017931:DNA Primers; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D017930:Genes, Reporter; D066298:In Vitro Techniques; D016133:Polymerase Chain Reaction
D017931:DNA Primers