A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors.
A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors.
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DOI：
10.1186/1472-6750-11-105
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发表时间：
2011-11-10
影响因子：
3.5
通讯作者：
Campbell RE
中科院分区：
文献类型：
--
作者：
Ibraheem A;Yap H;Ding Y;Campbell RE
基于分子内Förster共振能量转移(FRET)原理的基于荧光蛋白(FP)的生物传感器能够可视化活细胞中的各种生化事件。这些生物传感器的构建需要在FP的两种不同色调之间插入一个明智地选择的分子识别元件。当分子识别元件与感兴趣的分析物相互作用并经历构象变化时，由于FRET效率的变化，结构的比率发射被改变。这种生物传感器的灵敏度与比率发射的变化成正比，因此迫切需要各种方法来最大化现有生物传感器结构的比率变化，以增加其实用范围。为了加速改进的基于FRET的生物传感器的开发和优化，我们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体的方法。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的甲基化来展示这项技术。这项工作包括构建和筛选3个不同的文库：一个包含通过噬菌体展示分离的几个工程结合结构域的结构域文库；一个较低分辨率的连接库；以及一个较高分辨率的连接库。应用该文库筛选方法得到了一个优化的H3K27-三甲基化生物传感器，其发射比变化(66%)比最初构建的生物传感器(29%)提高了2.3倍。
Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Förster 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. 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. 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%).
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影响因子：
7.4
作者：
Campbell, Robert E.
通讯作者：
Campbell, Robert E.
影响因子：
10.5
作者：
Min, JR;Zhang, Y;Xu, RM
通讯作者：
Xu, RM
DOI：
10.1073/pnas.0712008105
发表时间：
2008-03-18
影响因子：
11.1
作者：
Hires, Samuel Andrew;Zhu, Yongling;Tsien, Roger Y.
通讯作者：
Tsien, Roger Y.
影响因子：
2.9
作者：
Gowher, H;Zhang, X;Jeltsch, A
通讯作者：
Jeltsch, A
DOI：
10.1038/nsb898
发表时间：
2003-03-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Manzur, KL;Farooq, A;Zhou, MM
通讯作者：
Zhou, MM