SORTING OF TRANSFECTED BACTERIA EXPRESSING GREEN FLUORESCENT PROTEIN

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. The goal of this project is to identify bacteria expressing soluble fluorescent protein after moleulcar evolution to improve protein folding and solubility for protein structure determination. Proteins whose structure is of interest will be genetically fused to a modified EGFP reporter and subjected to molecular evolution by DNA shuffling and error prone PCR. The resulting library will be transfeected into bacteria and expressed. Because the proper folding of the fusion construct is required for EGFP fluorescence, bacteria expressing soluble protein will be bright. Sorted bacteria are then cloned to isolate single variants of properly folded protein for crystallographic studies.