ANALYSIS AND SORTING OF BACTERIAL LIBRARY EXPRESSING GFP

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 GFP已被用作融合蛋白折叠和溶解的报告分子。以GFP为支架构建的文库由折叠良好的蛋白质和未折叠的蛋白质组成。折叠不好的GFP在选择过程中往往会给出背景。因此，我们希望用流式细胞术根据亮度将文库(108-109)分成三组。将从文库中选择结核或其他蛋白质的结合体。特定粘结剂的激发光谱和发射光谱将通过荧光法(Spex，Fluorolog)测定。在对粘合剂进行表征之后，将使用FacsCalibur测试它们在真核细胞上的有效性和分布。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. GFP has been used as reporter for folding and solubility of a fusion protein. Libraries made using GFP as scaffold consists of well folded as well as unfolded protein. GFP that is not folded well tends to give background during the selection. Therefore, we wish to sorted library (108-109) into three different groups based on the brightness using flow cytometry. Binders to TB or other proteins will be selected from the library. Excitation and emission spectra of specific binders will be determined by fluorimetry (Spex, Fluorolog). Following characterization of binders, their efficacy and distribution on/in eukoryotic cells will be tested using the FacsCalibur.