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Express Primer Tool for High Throughput Gene Cloning and Expression Poly Primer Design Tool Automated Cloning and Expression Methods Intranet Applications	High Throughput Molecular Biology The abundance of genomic sequence data from different organisms provides an opportunity to accelerate our understanding of protein structure and function. However, optimal utilization of this genomic information will require the development of high throughput methods for the generation of expression clones and the evaluation of protein function. Although high throughput screening methods are a standard component of most drug discovery platforms, many of the standard molecular protocols for gene cloning and expression analysis have only recently been implemented in a high throughput format. This is a consequence of the limited flexibility of an automated system as opposed to the classical methods for expression cloning. Alteration of manual cloning methods can be as simple as the purchase of a new cloning kit. However, substantial modification of an automation protocol can be expensive and demand significant amounts of time for rewriting and revalidation of protocols. The establishment of automated systems requires a more global approach for the evaluation and implementation of cloning and expression protocols. Protocols must be evaluated at the inception of the program with respect to their compatibility with other method protocols and for feasibility of implementation in an automated setting. In the Biosciences Division at Argonne National Laboratory, we have developed a series of methods for the automated generation of protein expression clones using a Beckman Coulter Core System. Each of these methods consists of a series of interlinked protocols representing liquid manipulations or incubations on various stations of the automation system. These protocols were initially developed to enable the automated generation of proteins for application in the field of structural genomics. However, the experience gained by implementation of these initial protocols provides a platform for extension of the system capabilities for application in other growth areas of high throughput molecular biology including site-specific mutagenesis, phage display, and protein interaction studies Molecular Biology Robot The Argonne Molecular Biology Robot System includes a number of stations (e.g., pipetting workstations, a heatblock, incubators…) that perform the equivalent of standard laboratory manipulations during an automation procedure. To allow for flexible programming of the system, the stations are treated in a modular manner. Thus, their activities will be kept independent and transportation of samples between stations will be performed such that the samples entering a station can arrive from and leave for any other station. This feature provides for maximum flexibility of method design and allows many programs to run concurrently. Applications High Throughput Strategies for Gene Cloning and Expression We have developed a series of automated protocols for the generation of bacterial expression clones using a robotic system designed for molecular biology procedures (Collart 2001) . These procedures were developed as a component of the high throughput Structural Genomics Pilot Center Program. Our approach uses the Ligation Independent Cloning (LIC) universal handle technology as an essential component of the method. This cloning approach allows the simultaneous insertion of a common PCR amplification product into multiple expression systems. Solubility or the expressed protein is determined by a plate-based assay system and confirmed by SDS-PAGE.    Click on the method names Automated Mutagenesis Methods The capability to generate an array of mutations in biofilm target proteins is enabled by application of automated mutagenesis protocols. The automated procedure we have developed is a PCR based approach that can be implemented using the established vector system for protein expression.
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