• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >A comparison of directed evolution approaches using the beta-glucuronidase model system.
【24h】

A comparison of directed evolution approaches using the beta-glucuronidase model system.

机译：使用β-葡萄糖醛酸苷酶模型系统的定向进化方法的比较。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.
机译：蛋白质工程师可以通过指导体外酶的进化来改变酶的特性。已经开发了许多产生分子多样性和鉴定改良克隆的方法，但是实验进化仍然是一门科学。我们以前使用DNA改组（性重组）和组织化学筛选来指导具有增强的β-半乳糖苷酶（BGAL）活性的大肠杆菌β-葡萄糖醛酸苷酶（GUS）变体的进化。在这里，我们采用相同的模型演化系统来测试其他几种技术的效率：递归随机诱变（无性），组合盒式诱变（高频重组）和通用的高通量微孔板筛选。在每项试验中都有特异性改变的GUS变体进化，但是诱变和筛选技术的不同组合影响了不同有益突变的固定。与以前使用的X-gal菌落筛选相比，新的微孔板筛选可识别出更广泛的突变。递归随机诱变基本上产生了无性种群，其中有益的突变使彼此绝种（克隆干扰）; DNA改组和组合盒诱变导致单个等位基因内有益突变的积累。这些结果解释了为什么重组方法通常会提高实验室进化的效率。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号