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Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step. Over the past ten years, protein engineering has established biocatalysis as a practical and environmentally friendly alternative to traditional forms of catalysis both in the laboratory and in industry. A new wave of engineered biocatalysts In 2001, a Review Article in Nature ( http://go.nature.com/t38uzr ) took stock of then-recent advances in biocatalysis — the use of enzymes or microbes to perform synthetic chemistry — and predicted steady development of the technology. In the intervening decade, biocatalysis has become a practical and environmentally friendly alternative to the use of transition-metal-based catalysts in chemical reactions. In a new Review, Bornscheuer et al . revisit the field. We are, they say, witnessing the third wave of biocatalysis, in which enzymes can be engineered with dramatic new activities. The authors highlight the success of biocatalysts in a number of applications, including the synthesis of important commodity chemicals and advanced pharmaceutical intermediates.

Separation and sorting of micron-sized particles has great importance in diagnostics, chemical and biological analyses, food and chemical processing and environmental assessment. By employing the unique characteristics of microscale flow phenomena, various techniques have been established for fast and accurate separation and sorting of microparticles in a continuous manner. The advancements in microfluidics enable sorting technologies that combine the benefits of continuous operation with small-sized scale suitable for manipulation and probing of individual particles or cells. Microfluidic sorting platforms require smaller sample volume, which has several benefits in terms of reduced cost of reagents, analysis time and less invasiveness to patients for sample extraction. Additionally, smaller size of device together with lower fabrication cost allows massive parallelization, which makes high-throughput sorting possible. Both passive and active separation and sorting techniques have been reported in literature. Passive techniques utilize the interaction between particles, flow field and the channel structure and do not require external fields. On the other hand, active techniques make use of external fields in various forms but offer better performance. This paper provides an extensive review of various passive and active separation techniques including basic theories and experimental details. The working principles are explained in detail, and performances of the devices are discussed.

Transcription activator–like effector nucleases (TALENs) are a new technology for modifying the genome at specific loci of interest. Hockemeyer et al . now demonstrate the utility of TALENs for gene targeting in human pluripotent stem cells. Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

The importance of developing new diagnostic and detection technologies for the growing number of clinical challenges is rising each year. Here, we present a concise, yet didactic review on a new diagnostics frontier based on the detection of disease-related volatile organic compounds (VOCs) by means of nanomaterial-based sensors. Nanomaterials are ideal for such sensor arrays because they are easily fabricated, chemically versatile and can be integrated into currently available sensing platforms. Following a general introduction, we provide a brief description of the VOC-related diseases concept. Then, we focus on detection of VOC-related diseases by selective and crossreactive sensing approaches, through chemical, optical and mechanical transducers incorporating the most important classes of nanomaterials. Selected examples of the integration of nanomaterials into selective sensors and crossreactive sensor arrays are given. We conclude with a brief discussion on the integration possibilities of different types of nanomaterials into sensor arrays, and the expected outcomes and limitations.

Nanoelectronic devices offer substantial potential for interrogating biological systems, although nearly all work has focused on planar device designs. We have overcome this limitation through synthetic integration of a nanoscale field-effect transistor (nanoFET) device at the tip of an acute-angle kinked silicon nanowire, where nanoscale connections are made by the arms of the kinked nanostructure, and remote multilayer interconnects allow three-dimensional (3D) probe presentation. The acute-angle probe geometry was designed and synthesized by controlling eis versus trans crystal conformations between adjacent kinks, and the nanoFET was localized through modulation doping. 3D nanoFET probes exhibited conductance and sensitivity in aqueous solution, independent of large mechanical deflections, and demonstrated high pH sensitivity. Additionally, 3D nanoprobes modified with phospholipid bilayers can enter single cells to allow robust recording of intracellular potentials.

Spintronics: diamonds make sense A type of natural impurity in diamond crystals, called a nitrogen-vacancy centre, has a unique, long-lived single electron spin state that can be controlled and detected optically. This property can be used to create 'spintronics' devices and has possible application in quantum information processing. Two groups this week describe the application of this technology to nanoscale magnetic resonance imaging. Maze et al . demonstrate magnetic sensing using coherent control of diamond spins. They show that in principle, precision measurements of nano-tesla magnetic fields are possible, corresponding roughly to the field of a single proton at a distance of 10 nm. Balasubramanian et al . demonstrate initial steps towards a sensitive, high-resolution imaging technique using diamond spins. They show that the location of single nitrogen-vacancy spins can be determined to 5-nm resolution. In an accompanying News & Views, Michael Romalis observes that a combination of these two techniques could lead to detection and imaging of individual nuclear spins, even the structure determination for a single molecule. And as both experiments were done at room temperature, biological applications of these methods can be anticipated. A naturally occurring impurity in diamond crystals, the nitrogen-vacancy centre, has been found to have a unique, long-lived single electron spin state that can be controlled and detected optically. An approach to magnetic sensing by coherent control of such diamond spins is demonstrated, and it is shown that precision measurements of nanoTesla magnetic fields are in principle possible. Detection of weak magnetic fields with nanoscale spatial resolution is an outstanding problem in the biological and physical sciences 1 , 2 , 3 , 4 , 5 . For example, at a distance of 10 nm, the spin of a single electron produces a magnetic field of about 1 μT, and the corresponding field from a single proton is a few nanoteslas. A sensor able to detect such magnetic fields with nanometre spatial resolution would enable powerful applications, ranging from the detection of magnetic resonance signals from individual electron or nuclear spins in complex biological molecules 5 , 6 to readout of classical or quantum bits of information encoded in an electron or nuclear spin memory 7 . Here we experimentally demonstrate an approach to such nanoscale magnetic sensing, using coherent manipulation of an individual electronic spin qubit associated with a nitrogen-vacancy impurity in diamond at room temperature 8 . Using an ultra-pure diamond sample, we achieve detection of 3 nT magnetic fields at kilohertz frequencies after 100 s of averaging. In addition, we demonstrate a sensitivity of 0.5 μT Hz -1/2 for a diamond nanocrystal with a diameter of 30 nm.

Analyzing the effects of multiple promoter motifs on gene expression can be a laborious process. Sharon et al . present a high-throughput method to measure the expression of thousands of designed yeast promoters in a single experiment and use it to reveal new features of transcriptional regulation. Despite extensive research, our understanding of the rules according to which cis -regulatory sequences are converted into gene expression is limited. We devised a method for obtaining parallel, highly accurate gene expression measurements from thousands of designed promoters and applied it to measure the effect of systematic changes in the location, number, orientation, affinity and organization of transcription-factor binding sites and nucleosome-disfavoring sequences. Our analyses reveal a clear relationship between expression and binding-site multiplicity, as well as dependencies of expression on the distance between transcription-factor binding sites and gene starts which are transcription-factor specific, including a striking ∼10-bp periodic relationship between gene expression and binding-site location. We show how this approach can measure transcription-factor sequence specificities and the sensitivity of transcription-factor sites to the surrounding sequence context, and compare the activity of 75 yeast transcription factors. Our method can be used to study both cis and trans effects of genotype on transcriptional, post-transcriptional and translational control.

We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including \"watermark\" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

The past decade has seen researchers develop and apply novel technologies for biomolecular detection, at times approaching hard limits imposed by physics and chemistry. In nearly all sensors, the transport of target molecules to the sensor can play as critical a role as the chemical reaction itself in governing binding kinetics, and ultimately performance. Yet rarely does an analysis of the interplay between diffusion, convection and reaction motivate experimental design or interpretation. Here we develop a physically intuitive and practical understanding of analyte transport for researchers who develop and employ biosensors based on surface capture. We explore the qualitatively distinct behaviors that result, develop rules of thumb to quickly determine how a given system will behave, and derive order-of-magnitude estimates for fundamental quantities of interest, such as fluxes, collection rates and equilibration times. We pay particular attention to collection limits for micro- and nanoscale sensors, and highlight unexplained discrepancies between reported values and theoretical limits.