• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>BMC Biotechnology >A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors
【24h】

A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors

机译：基于细菌菌落的筛选，用于遗传编码生物传感器中的最佳接头组合

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Background Fluorescent protein (FP)-based biosensors based on the principle of intramolecular F?rster resonance energy transfer ( FRET ) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility. Results To accelerate the development and optimization of improved FRET -based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli . We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library. Conclusion Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3 × improved relative to that of the initially constructed biosensor (29%).
机译：背景技术基于分子内Fster共振能量转移（FRET）原理的基于荧光蛋白（FP）的生物传感器，可以可视化活细胞中的各种生化事件。这些生物传感器的构造需要在FP的两种不同色调之间明智地选择分子识别元件进行基因插入。当分子识别元件与目标分析物相互作用并发生构象变化时，由于FRET效率的变化，构建体的比例发射也会发生变化。这种生物传感器的灵敏度与比例发射的变化成正比，因此迫切需要使现有生物传感器构造的比例变化最大化的方法，以增加其实用性。结果为了加快开发和优化基于FRET的生物传感器，我们已经开发了一种基于功能的高通量筛选大肠杆菌菌落中生物传感器变异的方法。我们已经通过优化生物传感器来检测组蛋白H3（H3K27）赖氨酸27的甲基化，证明了该技术。这项工作涉及3个不同文库的构建和筛选：一个域文库，其中包含通过噬菌体展示分离的几个工程结合域;低分辨率的链接器库;和更高分辨率的链接器库。结论该文库筛选方法的应用导致鉴定出优化的H3K27-三甲基化生物传感器，该传感器的发射率变化（66％）相对于最初构建的生物传感器（29％）提高了2.3倍。

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