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. 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.