coli, making the screening of the library more difficult if the substrate of the protein cannot be transported into the cell. However, proteins are usually expressed in cytoplasm in E. Because of its high transformation efficiencies, rapid growth rates, well-established manipulation approaches ( Crameri et al., 1996), Escherichia coli is generally employed as the host organism for library creation. This tool involves two crucial steps: the generation of a library containing sufficient gene variants, and high-throughput screening of library members with desired properties ( Wong et al., 2006 Packer and Liu, 2015). subtilis, facilitating directed evolution and expression optimization of target proteins.ĭirected evolution has been proved to be a powerful tool to improve the activity, stability, and substrate specificity of enzymes ( Wong et al., 2004, 2007 Roodveldt et al., 2005 Romero and Arnold, 2009 Goldsmith and Tawfik, 2017 Zeymer and Hilvert, 2018). Taken together, the present work provides a fast and efficient method to integrate epPCR products into the chromosome of B. The effectiveness of this method was confirmed by improving the activity of Methyl Parathion Hydrolase (MPH) toward chlorpyrifos and by enhancing the secretion level of MPH in B. The library generation process was accomplished within 1 day. A library containing 5.31 × 10 5 random mutants was constructed using per μg insertion construct, which is sufficient for directed evolution. The transformation efficiency of the insertion construct was improved through co-expressing homologous recombination-promoting protein NgAgo, raising the number of competent cells, and increasing the length of flanking regions. The epPCR product was integrated into the chromosome via homologous recombination after the insertion construct was transformed into the supercompetent cells of B. Specifically, the epPCR product was fused with flanking regions and antibiotic resistant marker using a PCR-based multimerization method, generating insertion construct. Here, a large library of random mutant was created by inserting error-prone PCR (epPCR) products to the chromosome of B. subtilis suffers problems of small library size, plasmid instability, and heterozygosity. However, the generation of a mutant library in B. Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Chinaīacillus subtilis is an attractive host for the directed evolution of the enzymes whose substrates cannot be transported across cell membrane.Bin Ye Yu Li Qing Tao Xiaoliang Yao Minggen Cheng Xin Yan *
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