title: Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli.
abstract: A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error-prone PCR or molecular shuffling, and a linear vector backbone was prepared by high-fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap-extension PCR with a pair of 5'-phosphorylated primers. Third, full-length linear plasmids with phosphorylated 5'-ends were self-ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high-efficiency transformation. Self-made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10(5) cfu/µg of the self-ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6-phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5-fold improved catalytic efficiency (kcat /Km ) on NAD(+) as compared to the wild-type. This protocol is DNA-sequence independent, and does not require restriction enzymes, special E. coli host, or labor-intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids.
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, USA.
authors.forename: Jae-Eung
authors.initials: JE
authors.lastname: Kim
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Huang
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Chun
authors.initials: C
authors.lastname: You
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, USA. ypzhang@vt.edu.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China. ypzhang@vt.edu.
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000378:metabolism; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D014169:Transformation, Bacterial; D000097573:Red Fluorescent Protein
chemical_list: D004247:DNA; D017931:DNA Primers; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: 6-phosphorogluconate dehydrogenase; Cherry fluorescent protein; Directed evolution; Moorella thermoacetica; Mutant library construction