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ARS Home » Research » Publications at this Location » Publication #177528


item Hughes, Stephen
item Mertens, Jeffrey
item Li, Xin Liang
item Bischoff, Kenneth
item Cotta, Michael

Submitted to: Journal of Laboratory Automation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/14/2005
Publication Date: 10/1/2005
Citation: Hughes, S.R., Riedmuller, S., Mertens, J.A., Li, X., Bischoff, K.M., Cotta, M.A., Farrelly, P. 2005. Development of a liquid handler component for a plasmid-based functional proteomic robotic workcell. Journal of the Association for Laboratory Automation. 10(5):287-300.

Interpretive Summary: Automated high-volume laboratory equipment is needed to rapidly produce large amounts of genes for improvement of bacterial and yeast strains to generate desired proteins, particularly optimized enzymes, in high yield for use in production of more efficient biocatalysts and high value bioproducts from underutilized agricultural materials. We have assembled the central component of this automated equipment and have shown it is capable of carrying out the required automated procedures consistently as programmed and of giving the desired products at high purity and yield. This will benefit companies and organizations that require equipment to rapidly produce and analyze large numbers of functionally active recombinant proteins, particularly commercially important enzymes, including agricultural and drug screening groups, identifying targets of insecticides and drugs, and those groups making new and improved strains of organisms for bioproduct and biocatalyst production.

Technical Abstract: A Hudson Control Group, Inc. Protein Express**TM robotic workcell to conduct plasmid-based functional proteomics is being developed for the optimization of protein open reading frames (ORF). The initial phase of this project is the design and assembly of a high-yield, high-speed liquid handler onto a Sias, Inc. Xantus® platform so that a workcell track component can be placed within the Xantus® gripper tool work area. The liquid handler is designed to produce plasmids using the Qiagen Turbo® plasmid preparation kit. This design allows processing of four 96-well plates in one run. The procedure eliminates disposable tips and provides an advanced wash system to prevent cross contamination. To evaluate the liquid handler operation, a mutagenized cellulase F ORF plasmid library was prepared from wild-type cellulase F**1,2 using a novel Invitrogen Gateway® cloning strategy. The average yield of plasmid was 3 ug per well from 1.35 mL of starting culture. The quality and quantity of plasmids prepared in this rapid mode on the liquid handler made possible the implementation of all workcell protocols: DNA sequencing, in vitro transcription/translation, transformation of BL21 DE3 expression bacteria, and transformation of yeast strains for scale up of expressed protein.