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Title: High-Throughput Fully Automated Construction of a Multiplex Library of Mutagenized Open Reading Frames for an Insecticidal Peptide Using a Plasmid-Based Functional Proteomic Robotic Workcell with Improved Vacuum System

item Hughes, Stephen
item Dowd, Patrick
item Hector, Ronald - Ron
item Mertens, Jeffrey
item Qureshi, Nasib
item Liu, Siqing
item Bischoff, Kenneth
item Li, Xin Liang
item Jackson Jr, John
item Rich, Joseph
item Cotta, Michael

Submitted to: Journal of the Association for Laboratory Automation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2007
Publication Date: 8/1/2007
Citation: Hughes, S.R., Dowd, P.F., Hector, R.E., Riedmuller, S.B., Bartolett, S., Mertens, J.A., Qureshi, N., Liu, S., Bischoff, K.M., Li, X., Jackson Jr, J.S., Sterner, D., Panavas, T., Cotta, M.A., Farrelly, P.J., Butt, T. 2007. High-throughput fully automated construction of a multiplex library of mutagenized open reading frames for an insecticidal peptide using a plasmid-based functional proteomic robotic workcell with improved vacuum system. Journal of Laboratory Automation. 12(4):202-212.

Interpretive Summary: Automated high-volume laboratory equipment is needed to rapidly produce large quantities of genes for improvement of bacterial and yeast strains to produce desired proteins in high yield for more efficient biocatalysts and high value bioproducts from underutilized agricultural materials. We have assembled a completely automated laboratory platform and shown it is capable of consistently performing the required programmed procedures and giving the desired products at high purity and yield. This will benefit companies and groups that require equipment to rapidly produce and analyze large numbers of functionally active optimized recombinant proteins, particularly commercially important enzymes and insecticidal peptides.

Technical Abstract: Robotic platforms are essential for the production and screening of large numbers of expression-ready plasmid sets used to develop optimized clones and improved microbial strains. Here we demonstrate a plasmid-based integrated workcell that was used to automate the molecular biology protocols including assembly of mutagenized gene sequences, purification of PCR amplicons, ligation of PCR products into vectors, transformation of competent Escherichia coli, plating of recovered transformants, and plasmid preparation. A library of genes encoding variants of wolf spider lycotoxin-1, a candidate bioinsecticide, was produced in the pENTR D TOPO vector using PCR mutagenesis in an amino acid scanning strategy to generate a complete set of mutations across the lycotoxin-1 gene. The plasmids were transformed without heat shock using rubidium chloride. Methods for recovery and growth of plasmid containing bacteria were designed to keep selective pressure but allow enough space for individual colony formation on a novel solid medium in a deepwell plate. The multiplexed wells in these plates were flooded with selective medium and used to inoculate plates for subsequent plasmid preparation. These protocols form the core of a fully automated molecular biology platform.