Nature Methods Contents: May 2014 Volume 11 pp 463 - 592

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Compare for yourself.  ReichertSPR.com/advantage Subscribe   Facebook   RSS   Recommend to library   Twitter   Advertisement Save 30%: Enhanced BD Horizon™ Brilliant Violet™ Reagents  Innovative new formulations of BD Horizon™ Brilliant Violet™ polymer conjugates are now available. Brighter than traditional dyes, these reagents can help you to resolve rare and dim cell populations, revealing nature's secrets with unprecedented clarity. Request a free sample or take 30% off purchases. bdbiosciences.com/go/brilliant   Advertisement They're recruiting scientists just like you! The Naturejobs Career Expo is coming to Boston, Massachusetts on May 20, 2014. Take advantage of this free opportunity to meet employers, attend workshops and further develop your career. Don't wait-register today!      In This Issue Top InThisIssue    Focus Top Focus on Synthetic Biology Contents Commentary Reviews Editorial Perspective Article Synthetic biology spans many disciplines and has many different goals but all rely on well-characterized basic tools. In this Focus experts discuss the current status of essential methods, going from DNA synthesis to genetic circuit design to whole genome assembly and how these tools can be deployed for applications from medical research to addressing the origin of life. Editorial Top Focus on Synthetic Biology Synthetic biology: back to the basics   p463 doi:10.1038/nmeth.2941 Realizing the ambitious goals of synthetic biology requires continued efforts in characterizing the foundations. This Month Top The author file: William Ja   p465 Vivien Marx doi:10.1038/nmeth.2927 Lessons from the volleyball court help to compare ways to measure how much flies eat. Points of significance: Nonparametric tests   pp467 - 468 Martin Krzywinski and Naomi Altman doi:10.1038/nmeth.2937 Nonparametric tests robustly compare skewed or ranked data. Research Highlights Top The big RNA picture Researchers sequence RNA directly within tissue. Chemical decaging Researchers design a chemical decaging strategy for studying protein function in cells. Embryonic matchmaking A nuclear-transfer technique informed by cell-cycle synchronization could simplify generation of therapeutic stem cells. Memoirs of a gut bacterium Engineered bacteria sense and record conditions in the gut. Background FRET Models are presented of background fluorescence resonance energy transfer (FRET) for non-interacting membrane proteins. A curveball for microscopists A curved beam of light provides microscopists with a new tool to overcome imaging challenges. Off-targets in RNAi screens A systematic analysis of genome-wide short interfering RNA (siRNA) screens in human cells identifies widespread off-target effects. Methods in Brief Tuning expression by numbers | Live-cell automated super-resolution microscopy | 5-formylcytosine-seq at single-base resolution | Cell-produced functional biomaterials Tools in Brief Computing single-cell trajectories | Monkey mutants with TALENs | Microbially produced ultrasound reporters | A small-molecule label for stem cells Methods JOBS of the week PhD fellowship The University of Hong Kong PhD Studentship: “Computational Modelling of Nucleation and Growth of Organic Crystals from Solution” Queen Mary University of London Director Institut Pasteur Postdoctoral Research Associate Washington University School of Medicine Postdoctoral Researcher Trinity College Dublin More Science jobs from Methods EVENT Statistical Methods for Functional Genomics 18th June 2014 Cold Spring Harbor, USA More science events from Technology Feature Top Cell-line authentication demystified   pp483 - 488 Vivien Marx doi:10.1038/nmeth.2932 Some researchers run and hide from the task of authenticating cell lines. A few simple steps save time and money. News and Views Top Discovering enhancers directly by activity   pp491 - 492 Ross C Hardison doi:10.1038/nmeth.2933 Two methods are used to identify cis-regulatory sequences by looking at their function. See also: Article by Murtha et al. | Article by Dickel et al. Commentary Top Focus on Synthetic Biology A synthetic approach to abiogenesis   pp495 - 498 James Attwater and Philipp Holliger doi:10.1038/nmeth.2893 Authors discuss how synthetic biology approaches could be applied to assemble synthetic quasibiological systems able to replicate and evolve, illuminating universal properties of life and the search for its origins. Reviews Top Focus on Synthetic Biology Large-scale de novo DNA synthesis: technologies and applications   pp499 - 507 Sriram Kosuri and George M Church doi:10.1038/nmeth.2918 This Review discusses large de novo DNA synthesis via oligos or arrays, describes gene assembly and error correction and considers applications for large-scale DNA synthesis. Focus on Synthetic Biology Principles of genetic circuit design   pp508 - 520 Jennifer A N Brophy and Christopher A Voigt doi:10.1038/nmeth.2926 This Review introduces tools to build transcriptional circuits and explains how the choice of different tools can affect circuit behavior and how its operation can be affected by the cellular host. Perspective Top Focus on Synthetic Biology Programming biological operating systems: genome design, assembly and activation   pp521 - 526 Daniel G Gibson doi:10.1038/nmeth.2894 This Perspective takes the reader through the important steps in bacterial genome assembly and activation and concludes with an outlook on how customized genomes may be achieved. Resource Top Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon   pp529 - 534 Christian Frøkjaer-Jensen, M Wayne Davis, Mihail Sarov, Jon Taylor, Stephane Flibotte et al. doi:10.1038/nmeth.2889 A minimal Mos transposon for integration of transgenes into the Caenorhabditis elegans genome expands the genetic toolbox in this model organism. Analysis Top Quantifying Drosophila food intake: comparative analysis of current methodology   pp535 - 540 Sonali A Deshpande, Gil B Carvalho, Ariadna Amador, Angela M Phillips, Sany Hoxha et al. doi:10.1038/nmeth.2899 This Analysis compares four commonly used assays to measure food intake in flies and identifies radioisotope-labeling and the capillary feeder (CAFE) as the most reproducible and sensitive. Brief Communications Top Light-sheet microscopy using an Airy beam   pp541 - 544 Tom Vettenburg, Heather I C Dalgarno, Jonathan Nylk, Clara Coll-Lladó, David E K Ferrier et al. doi:10.1038/nmeth.2922 Single-photon Airy beam excitation in light-sheet fluorescence microscopy combined with a simple deconvolution allows high-contrast imaging over a large field of view and minimal redundancy in illumination. Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers   pp545 - 548 Johan Hattne, Nathaniel Echols, Rosalie Tran, Jan Kern, Richard J Gildea et al. doi:10.1038/nmeth.2887 A computational approach and software tool, cctbx.xfel, enables the determination of accurate macromolecular structure factors using a relatively small number of serial femtosecond crystallography diffraction snapshots. Transcriptional profiling of cells sorted by RNA abundance   pp549 - 551 Sandy Klemm, Stefan Semrau, Kay Wiebrands, Dylan Mooijman, Dina A Faddah et al. doi:10.1038/nmeth.2910 Methods are described for preparing unbiased libraries for transcriptome profiling of cells sorted according to the abundance of a transcript of interest. Measuring similarity between dynamic ensembles of biomolecules   pp552 - 554 Shan Yang, Loic Salmon and Hashim M Al-Hashimi doi:10.1038/nmeth.2921 An approach for quantifying the similarity between dynamic ensembles of biomolecular structures is described and applied to RNA ensembles studied by nuclear magnetic resonance spectroscopy and molecular dynamics simulations. Single-molecule motions enable direct visualization of biomolecular interactions in solution   pp555 - 558 Quan Wang and W E Moerner doi:10.1038/nmeth.2882 Biomolecular interactions are directly detected and visualized in solution with a single-molecule method that measures time-dependent diffusion coefficient and mobility of electrokinetically trapped species. Advertisement Unlimited BioImaging: Chemiluminescence, Fluorescence, Colorimetric, Multiplex and More. The BioSpectrum Imaging System with its advanced capabilities enables a wide range of imaging applications. Unlimited in its ability to grow with your lab's developing life science research needs today and into the future. Learn More @ http://www.uvp.com/biospectrum.   Articles Top FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells   pp559 - 565 Matthew Murtha, Zeynep Tokcaer-Keskin, Zuojian Tang, Francesco Strino, Xi Chen et al. doi:10.1038/nmeth.2885 Screening of cells transduced with a lentivirus library harboring nucleosome-free regions of mammalian genomes identifies cis elements that regulate transcription. See also: News and Views by Hardison Function-based identification of mammalian enhancers using site-specific integration   pp566 - 571 Diane E Dickel, Yiwen Zhu, Alex S Nord, John N Wylie, Jennifer A Akiyama et al. doi:10.1038/nmeth.2886 Putative enhancer elements of a genomic region of interest are cloned in front of a reporter gene, integrated in a single site in mouse embryonic stem cells, and screened for enhancer activity using flow cytometry and high-throughput sequencing. See also: News and Views by Hardison Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein   pp572 - 578 Jun Chu, Russell D Haynes, Stéphane Y Corbel, Pengpeng Li, Emilio González-González et al. doi:10.1038/nmeth.2888 Three new red-excitable monomeric fluorescent proteins obtained by structure-guided mutagenesis of mNeptune are described in this work. The authors show the use of one of them, mCardinal, to visualize the differentiation of myoblasts into myocytes in living mice. Fluorescence nanoscopy by polarization modulation and polarization angle narrowing   pp579 - 584 Nour Hafi, Matthias Grunwald, Laura S van den Heuvel, Timo Aspelmeier, Jian-Hua Chen et al. doi:10.1038/nmeth.2919 Excitation using rotating polarized light and detection of periodic signals from rectangular nanoareas allows widef-ield super-resolution imaging of biological structures in cells and in tissue with reduced background. The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells   pp585 - 592 Julia Petschnigg, Bella Groisman, Max Kotlyar, Mikko Taipale, Yong Zheng et al. doi:10.1038/nmeth.2895 A method based on the split-ubiquitin assay monitors interactions between membrane proteins within human cells. 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