Page Banner

United States Department of Agriculture

Agricultural Research Service

Title: DEVELOPMENT OF A LIQUID HANDLER COMPONENT FOR A FUNCTIONAL PLASMID-BASED PROTEOMIC WORKCELL THAT GENERATES MULTIPLEX SAMPLES EXPRESSES IN YEAST)

Author
item Hughes, Stephen
item Riedmuller, Steven
item Bischoff, Kenneth
item Mertens, Jeffrey
item Li, Xin Liang
item Cotta, Michael
item Farrelly, Philip

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/5/2005
Publication Date: 2/5/2005
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.

Interpretive Summary:

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: 8/24/2016
Footer Content Back to Top of Page