ANALYSIS AND SORTING OF BACTERIAL LIBRARY EXPRESSING GFP

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 GFP已被用作融合蛋白的折叠和溶解性的报告基因。使用GFP作为支架制备的文库由良好折叠以及未折叠的蛋白质组成。没有很好折叠的GFP倾向于在选择期间提供背景。因此，我们希望使用流式细胞术基于亮度将文库（108-109）分选成三个不同的组。 将从文库中选择TB或其他蛋白质的结合剂。将通过荧光测定法（Spex，Fluorolog）测定特定结合剂的激发和发射光谱。在表征结合剂后，将使用FacsCalibur检测其在真核细胞上/中的功效和分布。

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.