Abstract
Laboratory Automation and High-Throughput Chemistry
Use of Robotics in High-Throughput DNA Sequencing
Robotics in life sciences has significantly improved in the last few years. In parallel, rapid advances in gene sequencing technology make it possible to sequence genes faster with more fidelity. Hence, it becomes important to revisit the advanced robotics and gene-sequencing technologies and examine them in the present years. Combination of these technologies in the current context will be more evolved and may be more appropriate for researchers. In this report, S. Keeny, reports automation protocols for DNA sequencing.
Until relatively recently, full sequencing of genes consisting of more than several exons was not considered practicable within a routine diagnostic context. As a result, many approaches to unknown mutation detection in a specific gene involved a mutation prescreening step to limit the amount of DNA sequencing required. Protocols to prescreen for mutations and to limit the amount of DNA sequencing may not localize every base change present and/or require considerable levels of manual intervention. Advances in technology, allied with careful protocol design, now permit direct DNA sequencing to be applied to larger areas of gene sequence, allowing unequivocal mutation identification in the area of a gene being analyzed. The protocol described uses robotic systems, allied to custom-designed PCR primers, to facilitate rapid DNA sequencing of multiple gene targets. The general approach is amenable to adaptation for use with multichannel pipettes (Keeney, S. Methods. Mol. Biol.
Automation Systems
Automated Microscopy for High-Content RNAi Screening
Fluorescence microscopy is one of the most powerful tools to investigate complex cellular processes such as cell division, cell motility, or intracellular trafficking. The availability of RNA interference (RNAi) technology and automated microscopy has opened the possibility to perform cellular imaging in functional genomics and other large-scale applications. Although imaging often dramatically increases the content of a screening assay, it poses new challenges to achieve accurate quantitative annotation and therefore needs to be carefully adjusted to the specific needs of individual screening applications. In this review, the authors discuss principles of assay design, large-scale RNAi, microscope automation, and computational data analysis; and they highlight strategies for imaging-based RNAi screening adapted to different library and assay designs (Conrad, C.; Gerlich, D. W. J. Cell. Biol.
Micro Reactor Technology
A Flow Bioreactor for Studying the Effects of Hemodynamic Forces on the Morphology and Rheology of Cylindrically Cultured Endothelial Cells
This study by Y. Reichenberg and Y. Lanir reports on the development and validation of an integrated experimental system for quantitative monitoring of the effects of vascular dynamic and static forces on endothelial cells (ECs), in terms of their morphological remodeling and rheological properties. The system consists of a microscope-based flow bioreactor that imposes controlled individual and combined hemodynamic forces on ECs cultured on the inner surface of cylindrical transparent substrate tubes. EC morphology is monitored by optical microscopy. Microrheological alterations are measured by optical magnetic twisting cytometry using ferromagnetic microbeads adherent to the EC cytoskeleton. System validation tests ascertain the capability for imposing controlled flow conditions and for real-time monitoring of morphological and rheological changes (J. Med. Eng. Technol.
Bioreactor Technology in Marine Microbiology: From Design to Future Application
In this review, the authors from Laboratory of Microbial Ecology and Technology of Ghent University report the application of bioreactor technology in marine microbiology focusing on energy generation and food production. Marine micro-organisms have been playing highly diverse roles over evolutionary time: they have defined the chemistry of the oceans and atmosphere. During the last decades, the bioreactors with novel designs have become important tools to study marine microbiology and ecology in terms of marine microorganism cultivation and deep-sea bioprocess characterization; unique biochemical product formation and intensification; and marine waste treatment and clean energy generation.
In this review, the authors briefly summarize the current status of the bioreactor technology applied in marine microbiology and the critical parameters to take into account during reactor design. Furthermore, the authors mention that from a look at the growing population and the pollution in the coastal areas of the world, it is urgent to find sustainable practices that beneficially stimulate both the economy and the natural environment. Here, Y. Zhang et al. suggest a few possibilities where innovative bioreactor technology can be applied to enhance energy generation and food production without harming the local marine ecosystem (Zhang, Y.; et al. Biotechnol. Adv.
Microfluidic Chip Technology
Wormometry on a Chip: Innovative Technologies for In Situ Analysis of Small Multicellular Organisms
Use of lab-on-a-chip (LOC) technologies at the molecular and cellular levels has been reported by several laboratories over the past decade. With ongoing development of chip technology, researchers realize that the application of this technology is not limited to existing studies, but can be expanded to other areas. One such expansion is the use of chip technology at an organism level.
In this report, the authors D. Wlodkowic et al. from University of Auckland, New Zealand report a new application for chip technology and present a new direction to the technology. The authors also report the use of microfluidics technology for cancer-related research in their previous publications. Small multicellular organisms such as nematodes, fruit flies, clawed frogs, and zebrafish are emerging models for an increasing number of biomedical and environmental studies. They offer substantial advantages over cell lines and isolated tissues, providing analysis under normal physiological milieu of the whole organism. Many bioassays performed on these alternative animal models mirror with a high level of accuracy those performed on inherently low-throughput, costly, and ethically controversial mammalian models of human disease.
Analysis of small model organisms in a high-throughput and high-content manner is, however, still a challenging task not easily susceptible to laboratory automation. In this context, recent advances in photonics, electronics, and material sciences have facilitated the emergence of miniaturized bioanalytical systems collectively known as LOC. These technologies combine microscale and nanoscale sciences, allowing the application of laminar fluid flow at ultralow volumes in spatially confined chip-based circuitry. LOC technologies are particularly advantageous for the development of a wide array of automated functionalities. The present work outlines the development of innovative miniaturized chip-based devices for the in situ analysis of small model organisms. The authors also introduce a new term “wormometry” to collectively distinguish these up-and-coming chip-based technologies that go far beyond the conventional meaning of the term “cytometry” (Wlodkowic, D.; et al. Cytometry. A.
Microfluidics-Enabled Phenotyping, Imaging, and Screening of Multicellular Organisms
Along similar lines as the preceding study by D. Wlodkowic et al., authors M. M. Crane et al. discuss the use of microfluidics technology for various applications. Some key advantages of the microfluidics technology, such as ability to control the microenvironment and the ease of automation, facilitate its use in diverse applications in a reliable manner.
This paper by M. M. Crane from Georgia Institute of Technology reviews the technologies that have been invented in the last few years on high-throughput phenotyping, imaging, screening, and related techniques using microfluidics. The review focuses on the technical challenges and how microfluidics can help to solve these existing problems, specifically discussing the applications of microfluidics to multicellular model organisms. The challenges facing this field include handling multicellular organisms in an efficient manner, controlling the microenvironment and precise manipulation of the local conditions to allow the phenotyping, screening, and imaging of the small animals.
Not only does microfluidics have the proper length scale for manipulating these biological entities, but automation has also been demonstrated with these systems, and more importantly the ability to deliver stimuli or alter biophysical/biochemical conditions to the biological entities with good spatial and temporal controls. In addition, integration with and interfacing to other hardware/software allows quantitative approaches. The authors include several successful examples of microfluidics solving these high-throughput problems. The article also highlights other applications that can be developed in the future (Crane, M. M.; et al. Lab Chip.
Microfluidics for Synthetic Biology From Design to Execution
This report by M. S. Ferry et al. discusses the importance and advantages of microfluidics. Aside from presenting the applicability of the technology with examples, the details of manufacturing the technology are also presented and are useful for researchers working in this field. With the expanding interest in cellular responses to dynamic environments, microfluidic devices have become important experimental platforms for biological research. Microfluidic “microchemostat” devices enable precise environmental control while capturing high-quality, single-cell gene expression data. For studies of population heterogeneity and gene expression noise, these abilities are crucial. Here, the authors describe the necessary steps for experimental microfluidics using devices created in their lab as examples.
The rational design of microchemostats and the tools available to predict their performance, analysis of critical parts of an example device, and focusing on the most important part of any microchemostat (the cell trap) are discussed. In the next phase, the authors present a method for generating on-chip dynamic environments using an integrated fluidic junction coupled to linear actuators. Their system relies on the simple modulation of hydrostatic pressure to alter the mixing ratio between two source reservoirs and they detail the software and hardware behind it.
To expand the throughput of microchemostat experiments, M. S. Ferry from the Department of Bioengineering of University of California describes how to build larger, parallel versions of simpler devices. To analyze the large amounts of data, they also discuss methods for automated cell tracking, focusing on the special problems presented by Saccharomyces cerevisiae cells. The manufacturing of microchemostats is described in complete detail from the photolithographic processing of the wafer to the final bonding of the polydimethylsiloxane chip to glass coverslip. Finally, the procedures for conducting Escherichia coli and S. cerevisiae microchemostat experiments are addressed (Ferry, M. S.; et al. Methods Enzymol.
High-Throughput Analytics
High-Fidelity Gene Synthesis by Retrieval of Sequence-Verified DNA Identified Using High-Throughput Pyrosequencing
One of the major challenges in next-generation sequencing (NGS) and producing synthetic DNA is the fidelity of the process. This report from Febit group addresses this issue and describes a platform that can significantly decrease the error rate of nucleotide sequences.
The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. The authors describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using NGS technology as a preparative tool. They demonstrate their method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared with the starting oligonucleotide pool generated by microarray. The authors use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized, and sorted in a single megacloner run, enabling constructive biology up to the megabase scale (Matzas, M. Nat. Biotechnol.
Advances in High-Throughput Recombinant Clone Production
New Ligation-Independent Cloning Vectors Compatible with a High-Throughput Platform for Parallel Construct Expression Evaluation Using Baculovirus-Infected Insect Cells
Ligation-independent cloning (LIC) is the most suitable method to clone and subclone genes in high-throughput manner. But before attempting the expensive cloning of thousands of genes, it is beneficial to understand the plasmid vector that will be used to express these genes. W. C. Brown et al. report their research on LIC vectors and their use in high-throughput cloning.
Biomedical research has undergone a major shift in emphasis over the past decade from characterizing the genomes of organisms to characterizing their proteomes. The high-throughput approaches that were successfully applied to sequencing of genomes, such as miniaturization and automation, have been adapted for high-throughput cloning and protein production. High-throughput platforms allow for a multiconstruct, multiparallel approach to expression optimization and construct evaluation. The authors describe a series of baculovirus transfer and expression vectors that contain LIC regions originally designed for use in high-throughput Escherichia coli expression evaluation.
These new vectors allow for parallel cloning of the same gene construct into a variety of baculovirus or E. coli expression vectors. A high-throughput platform for construct expression evaluation in baculovirus-infected insect cells is developed to use these vectors. Data from baculovirus infection expression trials for multiple constructs of two target protein systems relevant to the study of human diseases are presented. The target proteins exhibit a wide variation in behavior and illustrate the benefit of investigating multiple cell types, fusion partners, and secretion signals in optimization of constructs and conditions for eukaryotic protein production (Brown, W.C. Prot. Expr. Purif.