Cost-Effective 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

Abstract

Robotic platforms are essential for production of large numbers of expression-ready plasmid sets to develop optimized clones and improved microbial strains for crucial bioenergy applications and simultaneous high-value peptide expression. Here we demonstrate a plasmid-based integrated robotic workcell, modified with a motorized vacuum filtration system, for performing fully automated 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, plasmid preparation, cloning, and expression of optimized genes. A library of genes encoding variants of wolf spider lycotoxin-1, a candidate bioinsecticide, was produced using PCR mutagenesis in an amino acid scanning strategy to generate a complete set of mutations across the lycotoxin-1 gene. The improved vacuum filtration system permits automated purification of PCR products. Methods for recovery and growth of bacteria containing plasmids with PCR inserts allow individual colony formation on a novel solid medium in a deepwell plate. Inserts are cloned into a bacterial vector to verify expression. These protocols form the core of a fully automated molecular biology platform that reduces the cost and time required to perform all operations. (JALA 2007;12:202–12)