Journal of Laboratory Automation

This article describes a novel robotic system for protein expression and purification. This area has traditionally proved a bottleneck in pharmaceutical discovery owing to the difficulty and unpredictability of outcome in evaluating multiple sets of expression conditions that will yield biologically active protein in sufficient quantity to meet the demands of discovery teams. This position has been exacerbated by the requirement of structural biology groups to have access to 100 mg scale quantities of pure protein for early and complex structural studies. The Piccolo robotic system described here was able to perform multiple parallel induction, expression, and single stage protein purification of a target protein (6-His tagged β-galactosidase) using a model E. coli expression system. The Piccolo system monitored E. coli cell growth automatically and rescheduled tasks dynamically based on individual instances of cell growth within purpose built 24-well bioreactor units termed culture vessel blocks. On completion of protein expression, the system performed cell lysis and purification of the expressed β-galactosidase through Ni-NTA histidine affinity chromatography. We show that alterations in the induction and expression conditions used resulted in changes in the yield of expressed β-galactosidase.

In addition to the E. coli studies described here, the system is also applicable for other bacterial species and for eukaryotic cell culture applications.

The Piccolo robotic system has the capacity to allow exploration of hundreds of different combinations of expression variables within a working week thereby allowing rapid identification of optimal conditions for expressing the biologically active protein of interest. This strategy confers a significant increase in the parallel processing capability of protein expression groups and goes a long way to addressing the current protein expression bottleneck.

Embedded control programmable hardware and Windows-based Virtual Instruments (VI) simplify the design of automation in R&D laboratories. With minimal hardware and maximum support of software, the automation becomes more readily achievable (Jayapandian, J. Curr. Sci., 25 March 2006, 90(6) 765–70). A novel design is described for the control of a Siemens X-ray Diffractometer. The automation is achieved with custom-embedded hardware using an 89C2051 microcontroller that is compatible with a Universal Synchronous Bus port and a VI created using LabVIEW.

In response to the increasing demands to generate larger amounts of quality data faster, specifically in the area of RNA isolation to support gene expression assays, we have adopted several automated solutions for isolating total RNA from a variety of sample types, e.g., blood, cells, and tissue. For isolation from blood, collected in PAXgene (PreAnalytiX, Hombrechtickon, CH) tubes, we use a Qiagen BioRobot Universal System (Qiagen Inc-USA, Valencia, CA). For total RNA from cells, we have adapted a 96-well plate solid-phase extraction method using Promega SV96 (Promega, Inc., Madison, WI) reagents. Finally, if starting from tissues, we use the AutoGenPrep 245 (AutoGen Inc, Holliston, MA) robot to perform a modified acid guanidine phenol chloroform isolation. The use of the AutoGenPrep 245 is preceded by tissue homogenization, usually performed on the Qiagen TissueLyser.

Following total RNA isolation the RNA is quantified by QuantiT (Invitrogen Corporation, Carlsbad, CA) using a Biomek FX method that performs whole plate serial dilutions, and then creates a reaction plate with samples in duplicate. Finally, to check for genomic DNA contamination in the total RNA sample, a minus reverse transcription PCR assay is performed. In using these methods, we are able to increase our throughput without compromising data integrity.

Expression profiling and RNA interference (RNAi) studies require rapid and accurate ways to profile changes in gene expression. Such studies use methodologies for the isolation and subsequent analysis of mRNA because it is central to the transfer of genetic information within a cell. Quantitative reverse transcriptional PCR (RT-PCR) is routinely used to verify knockdown of gene expression mediated by RNAi, but its success is dependent upon the quality of the purified mRNA template. An automated system has been developed for the isolation and subsequent analysis of the mRNA. This system uses Sigma's SpyLine Poly A⁺ Capture Kit, a novel system for the rapid isolation of poly A+ mRNA from cultured mammalian cells for direct use in quantitative RT-PCR analysis. The automated method was used to identify effective RNAi gene knockdown. Results indicate that this approach has both the sensitivity and reproducibility necessary for measuring transcript levels following gene knockdown.

The dual-dye photometric technology used by Artel's Multichannel Verification System (MVS) overcomes challenges associated with determining the dispensed volume within each well of a 384-well microtiter plate. The MVS volume measurement approach (Bradshaw, J. T.; Knaide, T.; Rogers, A.; Curtis, R. Multichannel verification system (MVS): a dual dye ratiometric photometry system for performance verification of multichannel liquid delivery devices. Journal of the Association for Laboratory Automation 2005, 10, 35–42) has been extended to 384-well plates and volumes as low as 30 nL. The measurements collected by the MVS in less than 15 min provide both precision and accuracy values per individual channel. The system capabilities enable easy optimization of small-volume dispense protocols to minimize waste of valuable samples.

Amanual Mycobacterium avium subspecies paratuberculosis antibody enzyme linked immunosorbent assay (ELISA) (IDEXX Inc.) was fully automated using an automation workstation system (AWS). The IDEXX commercial kit is used to screen for exposure to the Johne's disease (JD) bacterium in cattle on a herd basis. Due to the large number of sera involved in this type of surveillance project, there is a need to increase the cost effectiveness and decrease the turn around time. The purpose of the work was to develop an automated ELISA (a-ELISA) for JD antibodies screening and examine the productivity advantages, quality assured performance characteristics, the laboratory information management (LIM) work, and the multitasking provisions under this format.

The AWS was assembled using a modular approach design. The high-throughput screening configuration consisted of a Beckman Coulter hybrid Biomek FX liquid-handling system integrated with a Molecular Devices Spectramax microplate reader, a Biotek ELx405 microplate washer, a Kendro Cytomat 6000 hotel, and an Axis PTZ Network camera. The integrated system function was programmed with an IBM computer using the SAMI software. This setup was housed in a Class 2 Biosafety Cabinet. This fully automated system provided flexibility to accommodate the multistep requirements of the immunoassay procedure. Complexity of the working steps was kept to a minimum by using one unique operation program. The complete robotic workstation with its peripheral equipment was simple to operate, robust, and easily maintained. These characteristics enabled the elimination of forced downtime due to human and/or machine failure. A 5 to 10 fold improvement in cost, test capacity, and productivity was achieved. With this system, one diagnostician is able to carry out over 1000 antibody screening tests a day. Based on the automated nature of the test, there is ample walk away time between the ELISA tests, enabling the technologist to multitask–performing other important duties such as sample receiving and preparation, test validation, result interpretation, information dissemination, and other related LIM work.

The performance characteristics–such as repeatability, reproducibility, optimal cutoff values, identity score, test sensitivity, and specificity were evaluated. These test validation results were based on data accumulated in an 8-year proficiency testing with the US National Veterinary Services Laboratory. Our laboratory test has received certification from the United States Department of Agriculture to perform serologic testing for JD since 1998. In 2000–2001, this test scope was accredited by the Standard Council of Canada. In 2004, this a-ELISA was validated according to the requirements of the ISO/IEC 17025 standard.