High efficiency and throughput system in directed evolution in vitro of reporter gene.
In vitro directed evolution, especially with DNA shuffling, is a powerful means in biological studies of protein structure and function, and consequently for industrial applications. Escherichia coli beta-glucuronidase (gusA) gene, a versatile and efficient reporter gene, was the model for studying in vitro directed evolution because of its stability, easy analysis of the enzyme properties and conveniently visible phenotype. We developed a high efficiency, throughput system for in vitro directed evolution using gusA reporter gene as the model. The system consisted mainly of three aspects: a prokaryotic expression vector pYPX251, an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy. The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E. coli. Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter. A library, which contained 100,000 individuals was screened by incubating ten filter papers with X-Glu. The polymerase chain reaction products of the gusA gene, the fragments of 50-100 bp, with high mutation rates were purified using a dialysis bag from 10% PAGE after electrophoresis. The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded. A GUS variant, named GUS-TR, was obtained through this system, which is significantly more resistant to high temperature than the wild type enzyme. GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100 degrees C for 30 min.
Xiong
Ai-Sheng
AS
Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, 2901 Beidi Road, Shanghai 201106, China.
Peng
Ri-He
RH
Liu
Jin-Ge
JG
Zhuang
Jing
J
Qiao
Yu-Shan
YS
Xu
Fang
F
Cai
Bing
B
Zhang
Zhen
Z
Chen
Jian-Min
JM
Yao
Quan-Hong
QH
D001709:Biotechnology / Q000379:methods; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D005966:Glucuronidase / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D005966:Glucuronidase