ANALYSIS AND SORTING OF BACTERIAL LIBRARY EXPRESSING GFP

Los Alamos Nat Secty-Los Alamos Nat Lab, Los Alamos NM

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.