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Cell-free biology is increasingly utilized for engineering biological systems, incorporating novel functionality, and circumventing many of the complications associated with cells. The central dogma describes the information flow in biology consisting of transcription and translation steps to decode genetic information. Aminoacyl tRNA synthetases (AARSs) and tRNAs are key components involved in translation and thus protein synthesis. This review provides information on AARSs and tRNA biochemistry, their role in the translation process, summarizes progress in cell-free engineering of tRNAs and AARSs, and discusses prospects and challenges lying ahead in cell-free engineering.

Although not employed in the clinic as of yet, microfluidic systems are likely to become a key technology for cancer diagnostics and prognosis. Microfluidic devices have been developed for the analysis of various biomarkers including circulating tumor cells, cell-free DNA, exosomes, and proteins, primarily in liquid biopsies such as serum, plasma, and whole blood, avoiding the need for tumor tissue biopsies. Here, we summarize microfluidic technological advances that are used in cancer diagnosis, prognosis, and to monitor its progression and recurrence, that will likely lead to personalized therapies. In some cases, integrated microfluidic technologies, coupled with biosensors, are proving to be more sensitive and precise in the detection of cancer biomarkers than conventional assays. Based on the current state-of-the-art and the rapid progress over the past decade, we also briefly discuss the next evolutionary steps that these technologies are likely to take.

The immune system is a complex network of specialized cells that work in concert to protect against invading pathogens and tissue damage. Imbalances in this network often result in excessive or absent immune responses leading to allergies, autoimmune diseases, and cancer. Many of the mechanisms and their regulation remain poorly understood. Immune cells are highly diverse, and an immune response is the result of a large number of molecular and cellular interactions both in time and space. Conventional bulk methods are often prone to miss important details by returning population-averaged results. There is a need in immunology to measure single cells and to study the dynamic interplay of immune cells with their environment. Advances in the fields of microsystems and microengineering gave rise to the field of microfluidics and its application to biology. Microfluidic systems enable the precise control of small volumes in the femto- to nanoliter range. By controlling device geometries, surface chemistry, and flow behavior, microfluidics can create a precisely defined microenvironment for single-cell studies with spatio-temporal control. These features are highly desirable for single-cell analysis and have made microfluidic devices useful tools for studying complex immune systems. In addition, microfluidic devices can achieve high-throughput measurements, enabling in-depth studies of complex systems. Microfluidics has been used in a large panel of biological applications, ranging from single-cell genomics, cell signaling and dynamics to cell-cell interaction and cell migration studies. In this review, we give an overview of state-of-the-art microfluidic techniques, their application to single-cell immunology, their advantages and drawbacks, and provide an outlook for the future of single-cell technologies in research and medicine.

Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner.

Although not employed in the clinic as of yet, microfluidic systems are likely to become a key technology for cancer diagnostics and prognosis. Microfluidic devices have been developed for the analysis of various biomarkers including circulating tumor cells, cell-free DNA, exosomes, and proteins, primarily in liquid biopsies such as serum, plasma, and whole blood, avoiding the need for tumor tissue biopsies. Here, we summarize microfluidic technological advances that are used in cancer diagnosis, prognosis, and to monitor its progression and recurrence, that will likely lead to personalized therapies. In some cases, integrated microfluidic technologies, coupled with biosensors, are proving to be more sensitive and precise in the detection of cancer biomarkers than conventional assays. Based on the current state-of-the-art and the rapid progress over the past decade, we also briefly discuss the next evolutionary steps that these technologies are likely to take.

The immune system is a complex network of specialized cells that work in concert to protect against invading pathogens and tissue damage. Imbalances in this network often result in excessive or absent immune responses leading to allergies, autoimmune diseases, and cancer. Many of the mechanisms and their regulation remain poorly understood. Immune cells are highly diverse, and an immune response is the result of a large number of molecular and cellular interactions both in time and space. Conventional bulk methods are often prone to miss important details by returning population-averaged results. There is a need in immunology to measure single cells and to study the dynamic interplay of immune cells with their environment. Advances in the fields of microsystems and microengineering gave rise to the field of microfluidics and its application to biology. Microfluidic systems enable the precise control of small volumes in the femto- to nanoliter range. By controlling device geometries, surface chemistry, and flow behavior, microfluidics can create a precisely defined microenvironment for single-cell studies with spatio-temporal control. These features are highly desirable for single-cell analysis and have made microfluidic devices useful tools for studying complex immune systems. In addition, microfluidic devices can achieve high-throughput measurements, enabling in-depth studies of complex systems. Microfluidics has been used in a large panel of biological applications, ranging from single-cell genomics, cell signaling and dynamics to cell-cell interaction and cell migration studies. In this review, we give an overview of state-of-the-art microfluidic techniques, their application to single-cell immunology, their advantages and drawbacks, and provide an outlook for the future of single-cell technologies in research and medicine.

Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner.

Contains fulltext : 209679.pdf (publisher's version ) (Open Access)

Contains fulltext : 209679.pdf (publisher's version ) (Open Access)

Contains fulltext : 209679.pdf (publisher's version ) (Open Access)

Mapping gene regulatory networks is a significant challenge in systems biology, yet only a few methods are currently capable of systems-level identification of transcription factors (TFs) that bind a specific regulatory element. We developed a microfluidic method for integrated systems-level interaction mapping of TF-DNA interactions, generating and interrogating an array of 423 full-length Drosophila TFs. With integrated systems-level interaction mapping, it is now possible to rapidly and quantitatively map gene regulatory networks of higher eukaryotes

High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We have developed a simple, yet powerful method that uses contact spotting to generate highdensity nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.

Mapping gene regulatory networks is a significant challenge in systems biology, yet only a few methods are currently capable of systems-level identification of transcription factors (TFs) thatbind a specific regulatory element. We developed a microfluidic method for integrated systems-level interaction mapping of TF–DNA interactions, generating and interrogating an array of 423 full-length Drosophila TFs. With integrated systems-level interaction mapping, it is now possible to rapidly and quantitatively map gene regulatory networks of higher eukaryotes.

Quantitative biology requires quantitative data. No high-throughput technologies exist capable of obtaining several hundred independent kinetic binding measurements in a single experiment. We present an integrated microfluidic device (k-MITOMI) for the simultaneous kinetic characterization of 768 biomolecular interactions. We applied k-MITOMI to the kinetic analysis of transcription factor (TF)—DNA interactions, measuring the detailed kinetic landscapes of the mouse TF Zif268, and the yeast TFs Tye7p, Yox1p, and Tbf1p. We demonstrated the integrated nature of k-MITOMI by expressing, purifying, and characterizing 27 additional yeast transcription factors in parallel on a single device. Overall, we obtained 2,388 association and dissociation curves of 223 unique molecular interactions with equilibrium dissociation constants ranging from 2×10^(-6)M to 2×10^(-9)M, and dissociation rate constants of approximately 6s^(-1) to 8.5×10^(-3)s^(-1). Association rate constants were uniform across 3 TF families, ranging from 3.7x10^6 M^(-1)s^(-1) to 9.6x10^7 M^(-1)s^(-1), and are well below the diffusion limit. We expect that k-MITOMI will contribute to our quantitative understanding of biological systems and accelerate the development and characterization of engineered systems.

Characterizing libraries of mutant proteins is a challenging task, but can lead to detailed functional insights on a specific protein, and general insights for families of proteins such as transcription factors. Challenges in mutant protein screening consist in synthesizing the necessary expression ready DNA constructs and transforming them into a suitable host for protein expression. Protein purification and characterization are also non-trivial tasks that are not easily scalable to hundreds or thousands of protein variants. Here we describe a method based on a high-throughput microfluidic platform to screen and characterize the binding profile of hundreds of transcription factor variants. DNA constructs are synthesized by a rapid two-step PCR approach without the need of cloning or transformation steps. All transcription factor mutants are expressed on-chip followed by characterization of their binding specificities against 64 different DNA target sequences. The current microfluidic platformcan synthesize and characterize up to 2400 protein–DNA pairs in parallel. The platform method is also generally applicable, allowing high-throughput functional studies of proteins.

Current gene synthesis methods allow the generation of long segments of dsDNA. We show that these techniques can be used to create synthetic regulatory elements and describe a method for the creation of completely defined, synthetic variants of the PHO5 promoter from the budding yeast Saccharomyces cerevisae. 128 promoters were assembled by high-temperature ligation, cloned into plasmids by isothermal assembly, maintained in E. coli, and consequently transformed into yeast by homologous recombination. Synthesis errors occurred at frequencies comparable to, or lower than those achieved with current gene synthesis methods. The promoter synthesis method reported here is robust, fast, and readily accessible. Synthetically engineered promoter libraries will be useful tools for dissecting the intricacies of promoter input-output functions, and may serve as tunable components for synthetic genetic networks.

During vertebrate embryogenesis, the rhythmic and sequential segmentation of the body axis is regulated by an oscillating genetic network termed the segmentation clock. We describe a new dynamic model for the core pace-making circuit of the zebrafish segmentation clock based on a systematic biochemical investigation of the network’s topology and precise measurements of somitogenesis dynamics in novel genetic mutants. We show that the core pace-making circuit consists of two distinct negative feedback loops, one with Her1 homodimers and the other with Her7:Hes6 heterodimers, operating in parallel. To explain the observed single and double mutant phenotypes of her1, her7, and hes6 mutant embryos in our dynamic model, we postulate that the availability and effective stability of the dimers with DNA binding activity is controlled in a "dimer cloud" that contains all possible dimeric combinations between the three factors. This feature of our model predicts that Hes6 protein levels should oscillate despite constant hes6 mRNA production, which we confirm experimentally using novel Hes6 antibodies. The control of the circuit’s dynamics by a population of dimers with and without DNA binding activity is a new principle for the segmentation clock and may be relevant to other biological clocks and transcriptional regulatory networks.

Specific binding of transcription factors (TFs) determines in a large part the connectivity of gene regulatory networks as well as the quantitative level of gene expression. A multiplicity of both experimental and computational methods is currently used to discover and characterize the underlying TF-DNA interactions. Experimental methods can be further subdivided into in vitro- and in vivo-based approaches, each accenting different aspects of TF-binding events. In this review we summarize the flexibility and performance of a selection of both types of experimental methods. In conclusion, we argue that a serial combination of methods with different throughput and data type constitutes an optimal experimental strategy.

DNA technologies such as cloning, DNA microarrays, and next generation sequencing have transformed the life sciences. Protein technologies on the other hand have not seen such explosive progress. This is mainly due to the inherent difficulty of working with proteins because of their manifold physical characteristics as opposed to the well behaved and well understood DNA polymer. Recent technological advancements have increased the throughput of protein biochemistry to levels where it is becoming of interest to systems biology. Here I review methods for high-throughput in situ synthesis and characterization of proteins and their integration with microfluidic devices. In the near future, the use of gene synthesis, microfluidic based protein synthesis and characterization will give rise to a resurgence of protein biochemistry in the current world of high-throughput genomics.

DNA technologies such as cloning, DNA microarrays, and next generation sequencing have transformed the life sciences. Protein technologies on the other hand have not seen such explosive progress. This is mainly due to the inherent difficulty of working with proteins because of their manifold physical characteristics as opposed to the well behaved and well understood DNA polymer. Recent technological advancements have increased the throughput of protein biochemistry to levels where it is becoming of interest to systems biology. Here I review methods for high-throughput in situ synthesis and characterization of proteins and their integration with microfluidic devices. In the near future, the use of gene synthesis, microfluidic based protein synthesis and characterization will give rise to a resurgence of protein biochemistry in the current world of high-throughput genomics.

Specific binding of transcription factors (TFs) determines in a large part the connectivity of gene regulatory networks as well as the quantitative level of gene expression. A multiplicity of both experimental and computational methods is currently used to discover and characterize the underlying TF-DNA interactions. Experimental methods can be further subdivided into in vitro- and in vivo-based approaches, each accenting different aspects of TF-binding events. In this review we summarize the flexibility and performance of a selection of both types of experimental methods. In conclusion, we argue that a serial combination of methods with different throughput and data type constitutes an optimal experimental strategy.

DNA technologies such as cloning, DNA microarrays, and next generation sequencing have transformed the life sciences. Protein technologies on the other hand have not seen such explosive progress. This is mainly due to the inherent difficulty of working with proteins because of their manifold physical characteristics as opposed to the well behaved and well understood DNA polymer. Recent technological advancements have increased the throughput of protein biochemistry to levels where it is becoming of interest to systems biology. Here I review methods for high-throughput in situ synthesis and characterization of proteins and their integration with microfluidic devices. In the near future, the use of gene synthesis, microfluidic based protein synthesis and characterization will give rise to a resurgence of protein biochemistry in the current world of high-throughput genomics.

The specific binding of transcription factors (TF) determines in a large part the connectivity of gene regulatory networks as well as the quantitative level of gene expression. A multiplicity of both experimental and computational methods is currently used to discover and characterize the underlying TF-DNA interactions. Experimental methods can be further subdivided into in vitro- and in vivo-based approaches, each accenting different aspects of TF binding events. In this review we summarize the flexibility and performance of a selection of both types of experimental methods. In conclusion, we argue that a serial combination of methods with different throughput and information content constitutes an optimal experimental strategy.

The tracking of cell populations in time-lapse microscopy images enables high-throughput spatiotemporal measurements of cell dynamics. In this paper, we present a new algorithm to simultaneously track many cells in crowded areas. The algorithm runs in real time and deals with thousands of cells. The main contribution of this paper is that the algorithm is able to maintain the spatiotemporal consistency of the tracks in crowded areas, even when the temporal resolution is coarse. We validate our approach in terms of its ability to track yeast cells.

Biology has always been a heavily technology limited field. Burgeoning fields such as systems biology require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfil many of the requirements put forth. Here I will discuss the various approaches employed to date for performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays.

Biology has always been a heavily technology limited field. Burgeoning fields such as systems biology require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfil many of the requirements put forth. Here I will discuss the various approaches employed to date for performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays.

Biology has always been a heavily technology limited field. Burgeoning fields such as systems biology require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfil many of the requirements put forth. Here I will discuss the various approaches employed to date for performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays.

Mapping gene regulatory networks is a significant challenge in systems biology, yet only a few methods are currently capable of systems-level identification of transcription factors (TFs) that bind a specific regulatory element. We developed a microfluidic method for integrated systems-level interaction mapping of TF-DNA interactions, generating and interrogating an array of 423 full-length Drosophila TFs. With integrated systems-level interaction mapping, it is now possible to rapidly and quantitatively map gene regulatory networks of higher eukaryotes