Evolving a two-photon bright green fluorescent protein for neuroscience

项目摘要 从活体模型生物的动态神经网络中收集数据的一个关键方法是 双光子显微镜和基因编码的荧光蛋白。有 需要优化荧光蛋白的双光子制度，因为一些可取的 特性（例如亮度）不从单光子优化转换。这 一个项目的目标是填补这一空白的定向演化的双光子明亮的绿色 荧光蛋白含有这种荧光蛋白的多个构建体将被 用于双光子成像。 定向进化将使用一种新的光学装置来实现， 双光子激发的荧光来自个体E.在琼脂平板上的大肠杆菌菌落。的 来自~8轮进化的最聪明的突变体将被结晶， 串联二聚体。二聚体将被掺入四种不同的构建体中：1） 与DREADD受体，2）与通道视紫红质，3）在组蛋白H2 B蛋白中 融合，和4）在Cre-FLEX开关表达载体中。所有结构将在 HEK细胞。

Project Summary A key method to gather data from dynamic neural networks in living model organisms is two-photon microscopy together with genetically-encoded fluorescent proteins. There is a need to optimize fluorescent proteins for the two-photon regime, as some desirable characteristics (e.g. brightness) do not translate from one-photon optimization. This project aims to fill that gap with the directed evolution of a two-photon bright green fluorescent protein. Multiple constructs containing this fluorescent protein will be created for use in two-photon imaging. The directed evolution will be implemented using a new optical setup that can collect two-photon excited fluorescence from individual E. coli colonies on an agar plate. The brightest mutants from ~8 rounds of evolution will be crystallized, and one will be made into a tandem dimer. The dimer will be incorporated into four different constructs: 1) with a DREADD receptor, 2) with a channelrhodopsin, 3) in a histone H2B protein fusion, and 4) in a Cre-FLEX-switch expression vector. All constructs will be tested in HEK cells.