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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL CATALYSTS TO PRODUCE FUEL ETHANOL AND VALUE ADDED PRODUCTS Title: Automated Strategy Using a Functional Proteomic Assay to Identify and Isolate Cellulase F Mutants with Improved Activity from Multiplexed Sets of Plasmid

Authors
item Hughes, Stephen
item Riedmuller, Steve - HUDSON CONTROL
item Mertens, Jeffrey
item Li, Xin Liang
item Qureshi, Nasib
item Farrelly, Philip - HUDSON CONTROL
item Cotta, Michael

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: January 13, 2006
Publication Date: January 13, 2006
Citation: Hughes, S.R., Riedmuller, S., Mertens, J.A., Li, X., Qureshi, N., Farrelly, P., Cotta, M.A. 2006. Automated strategy using a functional proteomic assay to identify and isolate cellulase F mutants with improved activity from multiplexed sets of plasmid [abstract]. PepTalk 2006. p. 10.

Technical Abstract: An automated strategy using a functional proteomic assay was designed for a plasmid-based functional proteomic robotic workcell to identify and isolate optimized cellulase F open reading frames from high-throughput multiplexed sets of mutagenized clones of the celF gene from the anaerobic fungus Orpinomyces PC-2 **1, 2. CelF mutants with improved activity were identified from an initial azo-carboxymethylcellulose plate screen of the mutants expressed from these clones, which were combined or "multiplexed" in sets of eight per well in a 96-well plate. The multiplexed wells containing these CelF mutants were linked back via bar coding to the wells containing the original multiplexed cultures stored in glycerol stock. Small samples from each well of the glycerol stocks were inoculated onto selective medium and the resulting colonies, each containing an individual clone, were picked robotically. This separated the identified multiplexed mixture of eight colonies into individual colonies, one or more giving rise to mutants with enhanced glucanase activity over the wild-type CelF open reading frame. Colony picking was performed into 24-well plates in quadruplicate giving 20 mL bacterial cultures containing more than 50 µg plasmid per well. These large-scale plasmid preparations were used in repeated automated cycles of in vitro transcription/translation to provide protein for automated azo-CMcellulose plate assays to evaluate the mutant CelF enzyme activity at various pHs and temperatures. Heat-stable clones with high activity at low pH were identified among the 768 insert-containing colonies tested.

Last Modified: 4/20/2014
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