Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group^1-3. JoVE video articles describing the method are available^1,4. The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group^5,6. The other two have recently become available and are included here for completeness. An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions.     

Automated template quantification for DNA sequencing facilities.

The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n=198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/microL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by >or=20% from the requested concentration (500 ng/microL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/microL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown.     

Improvement of Automatic In-Gel Digestion by in Situ Alkylation of Proteins

We have recently improved the automation of an in-gel digestion system, DigestPro 96, using in situ alkylation of proteins with acrylamide, conducted during one-dimensional (1D) SDS-PAGE. The improved method included the processes of destaining, dehydration, trypsin digestion, and extraction but excluded the reduction and alkylation steps following staining of proteins with CBB. The extracted peptide mixtures were directly loaded onto a micro C18 LC column of the mass spectrometer. The resultant spectra were processed with “Mascot” search engine to estimate the sequence coverage of the bovine serum albumin (BSA). The original method, designed for Laemmli 1D SDS gel applications, consisted of reduction and post-alkylation with iodoacetamide, which produced carboxyamidemethyl (CAM; –S–CH₂CONH₂) derivatives. The original method also included a desalting step essential for mass spectrometry, especially matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We compared the original and improved methods using BSA (3 pmol loaded to the gel, one third of digested peptide mixture injected into LC-MS). The original method yielded both CAM and propionicamide (PAM; –S–CH₂CH₂CONH₂) derivatives. The source of PAM derivatives is the unpolymerized acrylamide formed during electrophoresis. The sequence coverage of CAM derivatives of BSA by the original method was 10% with desalting and 19% without desalting. The sequence coverage of PAM derivative by the improved method was 32%. Our results clearly show the advantage of our improved automated in-gel digestion method for in situ PAM alkylated protein with respect to peptide recovery, compared with the original method with CAM post-alkylation.     

Rearing of larvae of Anopheles stephensi, using water replacement, purification and automated feeding

For rearing progenies of single females of Anopheles stephensi (Liston) (Diptera: Culicidae), a method is proposed using small trays with perforated side walls (called open trays) to allow for water replacement and purification using an aquatic plant Vallisneria spiralis. The performance of this method has been compared with that of rearing single progenies in trays without mesh side walls (closed trays). Effects of larval density and food quantity on rearing performance have been studied in both types of trays. In open trays the larvae developed about 2 days faster than in closed trays. Male larvae developed significantly faster (0.4 days) than females, but this difference was not significantly influenced by method of rearing or larval density. Adults from fast developing larvae were heavier than those emerging from slowly developing larvae from a single egg batch. Open trays gave higher larval survival than closed trays. A system has been developed to automatize larval feeding.

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Elevage des larves issues de femelles isolées d' Anopheles stephensi Liston (Dipt.; Culicidae), avec utilisation d'un système automatique d'alimentation et de renouvellement de l'eau

Résumé Une technique, utilisant des récipients avec des parois latérales perforées (baptisés récipients ouverts) permettant le renouvellement de l'eau et sa purification par une plante aquatique (Vallisneria piralis), est préconisée pour élever la descendance de femelles isolées de A. stephensi. Les performances de cette technique ont été comparées à l'élevage dans des récipients sans parois perforées (récipients clos). L'influence de la densité et de la quantité d'aliments a été examinée dans les deux cas. Dans les récipients ouverts les larves se développent plus vite (deux jours de moins); les mâles se développent plus vite que les femelles, mais cette différence (0,4 j) n'est significiativement pas influencée par les techniques d'élevage ou la densité. Pour une même ponte les adultes provenant des larves se développant le plus vite sont plus gros que ceux qui sont issus des larves les plus lentes. Le récipient ouvert donne une meilleure survie. Un système a été mis au point pour automatiser l'alimentation.

     

Methods and Results for Semi-automated Cloning Using Integrated Robotics

The Joint Center for Structural Genomics (JCSG) has emphasized automation and parallel processing approaches. Here, we describe automated methods used across the cloning process with results from JCSG projects. The protocols for PCR, restriction digests and ligations, as well as for gel electrophoresis and microtiter plate assays have all been automated. The system has the capacity to routinely process 384 clones a week. This throughput can adequately supply our expression and purification pipeline with expression-ready clones, including novel targets and truncations. The utility of our system is demonstrated by our results from three diverse projects. In summary, 94% of the PCR amplicons generated to date have been successfully cloned and verified by sequencing (83% of the total attempted targets). Our results demonstrate the capabilities of this robotic platform to provide an avenue to high-throughput cloning which requires little manpower and is rapid and cost-effective while providing insights for method optimization.     

Current good manufacturing practice in plant automation of biological production processes

The production of biologicals is subject to strict governmental regulations. These are drawn up in current good manufacturing practices (cGMP), a.o. by the U.S. Food and Drug Administration. To implement cGMP in a production facility, plant automation becomes an essential tool. For this purpose Manufacturing Execution Systems (MES) have been developed that control all operations inside a production facility. The introduction of these recipe-driven control systems that follow ISA S88 standards for batch processes has made it possible to implement cGMP regulations in the control strategy of biological production processes. Next to this, an MES offers additional features such as stock management, planning and routing tools, process-dependent control, implementation of software sensors and predictive models, application of historical data and on-line statistical techniques for trend analysis and detection of instrumentation failures. This paper focuses on the development of new production strategies in which cGMP guidelines are an essential part.     

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays

The mouse bone marrow micronucleus assay is anin vivo test commonly used in the pharmaceutical industry to evaluate the genotoxic potential of new compounds. The test detects agent-induced chromosomal damage or damage of the mitotic spindle apparatus. In this paper the state-of-the-art in automated rodent micronucleus evaluation using computerized image analyis in combination with high-quality slides obtained by the cellulose column fractionation technique is reviewed. The latter allows the effective removal of nucleated cells from rodent bone marrow. It has been found that automatic micronucleus scoring with the Leitz MIAC image analyzer is substantially faster than labor-intensive manual analysis. Automatic scoring can be performed overnight for up to 16 slides. We have been successfully using automatic micronucleus analysis for the testing of new pharmaceutical drugs for more than 3 years.Abbreviations MNE NCE containing micronuclei - MPE PCE containing micronuclei - NCE normochromatic erythrocyte - PCE polychromatic erythrocyte deceased on 25 May 1994     

Animal board invited review: precision livestock farming for dairy cows with a focus on oestrus detection

Dairy cows are high value farm animals requiring careful management to achieve the best results. Since the advent of robotic and high throughput milking, the traditional few minutes available for individual human attention daily has disappeared and new automated technologies have been applied to improve monitoring of dairy cow production, nutrition, fertility, health and welfare. Cows milked by robots must meet legal requirements to detect healthy milk. This review focuses on emerging technical approaches in those areas of high cost to the farmer (fertility, metabolic disorders, mastitis, lameness and calving). The availability of low cost tri-axial accelerometers and wireless telemetry has allowed accurate models of behaviour to be developed and sometimes combined with rumination activity detected by acoustic sensors to detect oestrus; other measures (milk and skin temperature, electronic noses, milk yield) have been abandoned. In-line biosensors have been developed to detect markers for ovulation, pregnancy, lactose, mastitis and metabolic changes. Wireless telemetry has been applied to develop boluses for monitoring the rumen pH and temperature to detect metabolic disorders. Udder health requires a multisensing approach due to the varying inflammatory responses collectively described as mastitis. Lameness can be detected by walk over weigh cells, but also by various types of video image analysis and speed measurement. Prediction and detection of calving time is an area of active research mostly focused on behavioural change.