Progress in the construction and screening of random mutation library / 生物工程学报

Directed evolution is a cyclic process that alternates between constructing different genes and screening functional gene variants. It has been widely used in optimization and analysis of DNA sequence, gene function and protein structure. It includes random gene libraries construction, gene expression in suitable hosts and mutant libraries screening. The key to construct gene library is the storage capacity and mutation diversity, to screen is high sensitivity and high throughput. This review discusses the latest advances in directed evolution. These new technologies greatly accelerate and simplify the traditional directional evolution process and promote the development of directed evolution.

Construction and quality analysis of phage display library for random mutagenesis of camel nanobody / 国际生物医学工程杂志

Objective To construct phage display antibody library of artificial mutation to compare with the sequence of the natural phage display antibody library. To scientifically evaluate the quality of the artificial mutation of phage display library, and provide some references for the further transformation of the nanobody. Methods Using random mutation method, NNY fixed-point santuration mutation was performed on combine the follicle-stimulating hormone receptor (FSHR) of human nanobody. The mutant DNA sequence was connected to the vector pMECS to construct the phage display library of VHH06-CDR3 random mutation. By sequencing and analysis of DNA sequences, the diversity of the library and the amino acid distribution of CDR3 were compared between mutation library and the immune library of FSHR. The degree of enrichment of cloning was determined by six rounds of affinity screening. Results According to the NNY mutation rule ,the CDR3 regions with 16 amino acids by random mutations was synthesized and the VHH-CDR3 random mutant phage display library was constructed . The phage display library of VHH06-CDR3 random mutant size was 7.36×108 cfu/ml. Polyclonal and monoclonal phage ELISA showed that after six rounds of screening, the output phage and the combination of FSHR showed obvious enrichment, but there was no clone combined with FSHR. Conclusions Although the VHH06-CDR3 mutant phage display library has sequence diversity, it is not conducive to obtaining target antibodies in affinity screening due to the lack of functional diversity of CDR3.

Enhancement of cellulolytic enzymes and xylanase production via classical mutational techniques under solid-state fermentation condition

Aims: High cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass (LB). The present study aims at developing a local cellulolytic fungal strain through random mutagenesis coupled with the feasibility of solid-state fermentation (SSF) by utilizing agricultural wastes such as oil palm frond (OPF) as the substrate. Methodology and results: Out of 95 wild isolates tested, native fungal strain Aspergillus niger, designated DWA8 was isolated as the top enzymatic secretor. For quantitative enzyme analysis, SSF was conducted using 1x106 spore/mL inoculated onto 5 g of ground OPF, incubated at room temperature for 7 days, with 70% moisture content and an initial medium pH of 7. Random mutagenesis has always been tempting in the enhancement of enzyme production. In this work, the compounded treatment of microwave, ultraviolet (UVC) and Ethyl Methanesulfonate (EMS) have generated an Aspergillus niger MUE3.06 mutant with an overall increase of 114% in CMCase activity, approximately 70% in FPase and Xylanase activity respectively compared with the parental DWA8 strain. Thus this finding is capable to be fully developed as an established mutational scheme to create highly productive filamentous fungus in a cheap, simple and sustainable way. Conclusion, significance and impact of study: It was the first attempt to explore the combine effect of the three popular mutagens upon cellulases and xylanases. It is believed that more diversified of mutagen types induce more diversified mutation pattern (with instructive planning), which is very desirable in creating new enzymes with novel abilities.

The condition for error-prone PCR induced mutation / 第二军医大学学报

Objective: To evaluate the effectiveness of random mutagenesis via mutator E.coli strain and error prone PCR. Methods: A ScFv containing phagemid was transformed into mutator strain XL1 Red and subjected to 7 overnight growth phases with 1/100 dilution of each phase. DNA was extracted and sequenced. The ScFv gene was also subjected to PCR mutagenesis. Mutation effects of Mg 2+ concentration, alteration of individual dNTP amounts and addition of dITP were investigated. Results: The mutation rate of 7 growth phases (about 50 cell cycles) in XL1 Red was less than 0.1%. In error prone PCR, higher concentration of Mg 2+ increased the mutation rate. Increased content of dTTP and dCTP had better effect than that of dATP and dGTP. Addition of dITP plus low concentration dATP or dGTP caused lower mutation rate. More than 2% mutation could be reached by 2 rounds PCR containing 5 mmol/L MgCl 2, 0.5 mmol/L MnCl 2, 1 mmol/L dTTP and 1 mmol/L dCTP. But the mutation showed obvious base bias with most substitutions at A/T positions and a prevalence of transition over transversion. Conclusion: Random mutagenesis in mutator strain has too low mutation rate for antibody affinity maturation. A more than 2% mutation rate can be obtained by error prone PCR, which is suitable for constructing mutated antibody libraries, but the base bias of error prone PCR should be considered.