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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: Development of a Liquid Handler Component for a Functional Plasmid-Based Proteomic Workcell That Generates Multiplex Samples Expresses in Yeast

Authors
item Hughes, Stephen
item Riedmuller, Steven - HUDSON CONTROL
item Bischoff, Kenneth
item Mertens, Jeffrey
item Li, Xin Liang
item Cotta, Michael
item Farrelly, Philip - HUDSON CONTROL

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 5, 2005
Publication Date: February 5, 2005
Citation: Hughes, S.R., Riedmuller, S.B., Bischoff, K.M., Mertens, J.A., Li, X., Cotta, M.A., Farrelly, P.J. 2005. Development of a liquid handler component for a functional plasmid-based proteomic workcell that generates multiplex samples expresses in yeast [abstract]. Association for Laboratory Automation, LabAutomation 2005. Poster WP128.

Technical Abstract: We are assembling a workcell to conduct plasmid-based functional proteomics for the optimization of protein open reading frames (ORF) and for screening of improved yeast and bacterial strains. For this project a liquid handler was designed and assembled onto the Sias Xantus platform so that a Hudson Control Group track component could be placed within the Xantus gripper tool work area for generating plasmids using the Qiagen Turbo plasmid preparation kit, which gives four 96-well plates of plasmids in one run. In generating this set of plasmids, 20 ug of 96 different clones or up to 5 ug of 384 individual clones can be obtained. Methods were developed for the workcell using the plasmids generated to evaluate their quality. As an example using these methods, a mutgenesis strategy was designed for the cellulase F ORF from Orpinomyces, and an optimized mutant was identified. The plasmids obtained were used in sequencing reactions, in vitro transcription/translation, and transformation of BL21 DE3 expression bacteria, as well as transformation of yeast strains for scale up of expressed protein and for AZO-cellulose plate assays. The liquid handler was found to provide sufficient plasmid DNA for all methods to be used on the workcell including a routine for multiplex sets of plasmid that were expressed in vitro and in vivo.

Last Modified: 7/30/2014
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