ALBERT

Description: In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis -regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila . We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F 1 generation.

Description: Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ~40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

Description: We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9–1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p -azido- l -phenylalanine (pAzF) or p -propargyloxy- l -phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50–88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA opt ) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.

Description: Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.

Description: A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.

Description: We describe a novel cloning method termed SLiCE (Seamless L i gation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

Description: Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.

Description: A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.

Description: Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli . This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.

Description: To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11 , located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a ‘landing pad’ cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson’s disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.

Description: Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli , usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.

Description: The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc R ) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc R . A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.

Description: DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (~1 kb), pathway engineering (~10 kb) or synthetic genomes (〉100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at 〈$1 per sample.

Description: Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP / attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.

Description: A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z 3 EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z 3 EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z 3 EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.

Description: We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii . Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus . Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.

Description: The increasing interest in genetic manipulation of bacterial host metabolic pathways for protein or small molecule production has led to a need to add new genes to a chromosome quickly and easily without leaving behind a selectable marker. The present report describes a vector and four-day procedure that enable site-specific chromosomal insertion of cloned genes in a context insulated from external transcription, usable once in a construction series. The use of rhamnose-inducible transcription from rhaBp allows regulation of the inserted genes independently of the commonly used IPTG and arabinose strategies. Using lacZ as a reporter, we first show that expression from the rhamnose promoter is tightly regulatable, exhibiting very low leakage of background expression compared with background, and moderate rhamnose-induced expression compared with IPTG-induced expression from lacp . Second, the expression of a DNA methyltransferase was used to show that rhamnose regulation yielded on-off expression of this enzyme, such that a resident high-copy plasmid was either fully sensitive or fully resistant to isoschizomer restriction enzyme cleavage. In both cases, growth medium manipulation allows intermediate levels of expression. The vehicle can also be adapted as an ORF-cloning vector.

Description: The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3'-〉5' exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by 〉16-fold, yielding a final error rate of ~1 in 8700 bp.

Description: Passive interferometry technology is based on the relation between the reflection and the transmission responses of the subsurface. The transmission response can be received at surface in the presence of the ambient noise source in the subsurface with the cross-correlation (CC) or multidimensional deconvolution methods. We investigate the feasibility of electromagnetic (EM) wave passive interferometry with CC method. We design a 2-D finite-difference time domain (FDTD) algorithm to simulate the long-duration ground penetrating radar (GPR) measurements with random distribution of passive EM sources. The noise sources have random duration time, waveform and spatial distribution. We test the FDTD GPR passive interferometry code with above source characteristics and apply the method to light non-aqueous phase liquid (LNAPL) monitoring. Based on the model simulation data, by using common midpoint velocity analysis and normal move out correction to process the interferometry retrieve record, we can accurately obtain the dynamic changing characteristics of the target's permittivity. The LNAPL dynamic leakage model can be imaged as well. The synthetic results demonstrate that the GPR passive interferometry is feasible in subsurface LNAPL monitoring. Our work provides a foundation for a passive interferometry field application using GPR.

Description: 3-D electrical resistivity surveys and inversion models are required to accurately resolve structures in areas with very complex geology where 2-D models might suffer from artefacts. Many 3-D surveys use a grid where the number of electrodes along one direction ( x ) is much greater than in the perpendicular direction ( y ). Frequently, due to limitations in the number of independent electrodes in the multi-electrode system, the surveys use a roll-along system with a small number of parallel survey lines aligned along the x -direction. The ‘Compare R’ array optimization method previously used for 2-D surveys is adapted for such 3-D surveys. Offset versions of the inline arrays used in 2-D surveys are included in the number of possible arrays (the comprehensive data set) to improve the sensitivity to structures in between the lines. The array geometric factor and its relative error are used to filter out potentially unstable arrays in the construction of the comprehensive data set. Comparisons of the conventional (consisting of dipole-dipole and Wenner–Schlumberger arrays) and optimized arrays are made using a synthetic model and experimental measurements in a tank. The tests show that structures located between the lines are better resolved with the optimized arrays. The optimized arrays also have significantly better depth resolution compared to the conventional arrays.

Description: We apply a reversible-jump Markov chain Monte Carlo method to sample the Bayesian posterior model probability density function of 2-D seafloor resistivity as constrained by marine controlled source electromagnetic data. This density function of earth models conveys information on which parts of the model space are illuminated by the data. Whereas conventional gradient-based inversion approaches require subjective regularization choices to stabilize this highly non-linear and non-unique inverse problem and provide only a single solution with no model uncertainty information, the method we use entirely avoids model regularization. The result of our approach is an ensemble of models that can be visualized and queried to provide meaningful information about the sensitivity of the data to the subsurface, and the level of resolution of model parameters. We represent models in 2-D using a Voronoi cell parametrization. To make the 2-D problem practical, we use a source–receiver common midpoint approximation with 1-D forward modelling. Our algorithm is transdimensional and self-parametrizing where the number of resistivity cells within a 2-D depth section is variable, as are their positions and geometries. Two synthetic studies demonstrate the algorithm's use in the appraisal of a thin, segmented, resistive reservoir which makes for a challenging exploration target. As a demonstration example, we apply our method to survey data collected over the Scarborough gas field on the Northwest Australian shelf.

Description: Compressional and shear wave seismic measurements were performed in an old railway tunnel and in galleries excavated in a 250-m-thick Toarcian claystone formation in the Tournemire experimental station (France). Three component (3C) geophones and three orthogonal orientations of the vibroseismic force source were used. Additionally, vertical seismic profiling (VSP) measurements were recorded with a 3C borehole geophone, a hydrophone and a microphone in a 159 m deep borehole (ID180) in the tunnel. The seismic data show that Toarcian claystone has strong transverse isotropy (TI) with a vertical symmetry axis. The qP , SH and qSV wave propagation velocities in horizontal directions—the plane of isotropy of the TI medium—are measured as 3550, 1850 and 1290 m s –1 , respectively. The zero-offset VSP reveals that only one shear wave propagates in the vertical (depth) direction and the P - and S -wave velocities are 3100 and 1375 m s –1 , respectively. Four elastic moduli of the TI medium are determined from the seismic velocities and from the bulk density of 2.53 g cm –3 : c 11 = 31.9 GPa, c 33 = 24.3 GPa, c 44 = 4.5 GPa and c 66 = 8.7 GPa. A walkaway VSP with the borehole geophone at 50 m depth in borehole ID180 and shot points in the galleries leads to oblique seismic ray paths which allow us to determine the fifth elastic modulus of the TI medium to c 13 = 16 GPa. The tube wave recorded by a hydrophone in the water filled lower part of the borehole propagates with 1350 m s –1 , which confirms the estimate of the elastic constant c 66 . The analysis of body wave and surface wave data from a seismic experiment in Galerie Est shows reflections from several fracture zones in the gallery floor. The thickness of the excavation damaged zone (EDZ) in the floor of Galerie Est is estimated to 0.7 m.

Description: The Tonga-Kermadec forearc is deforming in response to on-going subduction of the Pacific Plate beneath the Indo-Australian Plate. Previous research has focussed on the structural development of the forearc where large bathymetric features such as the Hikurangi Plateau and Louisville Ridge seamount chain are being subducted. Consequently, knowledge of the ‘background’ forearc in regions of normal plate convergence is limited. We report on an ~250-km-long multichannel seismic reflection profile that was shot perpendicular to the Tonga-Kermadec trench at ~28°S to determine the lateral and temporal variations in the structure, stratigraphy and deformation of the Kermadec forearc resulting solely from Pacific Plate subduction. Interpretation of the seismic profile, in conjunction with regional swath bathymetry data, shows that the Pacific Plate exhibits horst and graben structures that accommodate bending-induced extensional stresses, generated as the trenchward dip of the crust increases. Trench infill is also much thicker than expected at 1 km which, we propose, results from increased sediment flux into and along the trench. Pervasive normal faulting of the mid-trench slope most likely accommodates the majority of the observed forearc extension in response to basal subduction erosion, and a structural high is located between the mid- and upper-trench slopes. We interpret this high as representing a dense and most likely structurally robust region of crust lying beneath this region. Sediment of the upper-trench slope documents depositional hiatuses and on-going uplift of the arc. Strong along-arc currents appear to erode the Kermadec volcanic arc and distribute this sediment to the surrounding basins, while currents over the forearc redistribute deposits as sediment waves. Minor uplift of the transitional Kermadec forearc, observed just to the north of the profile, appears to relate to an underlying structural trend as well as subduction of the Louisville Ridge seamount chain 250 km to the north. Relative uplift of the Kermadec arc is observed from changes in the tilt of upper-trench slope deposits and extensional faulting of the basement immediately surrounding the Louisville Ridge.

Description: The precise control of gene expression is essential in basic biological research as well as in biotechnological applications. Most regulated systems available in yeast enable only the overexpression of the target gene, excluding the possibility of intermediate or weak expression. Moreover, these systems are frequently toxic or depend on growth conditions. We constructed a heterologous transcription factor that overcomes these limitations. Our system is a fusion of the bacterial LexA DNA-binding protein, the human estrogen receptor (ER) and an activation domain (AD). The activity of this chimera, called LexA-ER-AD, is tightly regulated by the hormone β-estradiol. The selection of the AD proved to be crucial to avoid toxic effects and to define the range of activity that can be precisely tuned with β-estradiol. As our system is based on a heterologous DNA-binding domain, induction in different metabolic contexts is possible. Additionally, by controlling the number of LexA-binding sites in the target promoter, one can scale the expression levels up or down. Overall, our LexA-ER-AD system is a valuable tool to precisely control gene expression in different experimental contexts without toxic side effects.

Description: Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

Description: Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.

Description: New methods are required to combine the information contained in the passive electrical and seismic signals to detect, localize and monitor hydromechanical disturbances in porous media. We propose a field experiment showing how passive seismic and electrical data can be combined together to detect a preferential flow path associated with internal erosion in a Earth dam. Continuous passive seismic and electrical (self-potential) monitoring data were recorded during a 7-d full-scale levee (earthen embankment) failure test, conducted in Booneschans, Netherlands in 2012. Spatially coherent acoustic emissions events and the development of a self-potential anomaly, associated with induced concentrated seepage and internal erosion phenomena, were identified and imaged near the downstream toe of the embankment, in an area that subsequently developed a series of concentrated water flows and sand boils, and where liquefaction of the embankment toe eventually developed. We present a new 4-D grid-search algorithm for acoustic emissions localization in both time and space, and the application of the localization results to add spatially varying constraints to time-lapse 3-D modelling of self-potential data in the terms of source current localization. Seismic signal localization results are utilized to build a set of time-invariant yet spatially varying model weights used for the inversion of the self-potential data. Results from the combination of these two passive techniques show results that are more consistent in terms of focused ground water flow with respect to visual observation on the embankment. This approach to geophysical monitoring of earthen embankments provides an improved approach for early detection and imaging of the development of embankment defects associated with concentrated seepage and internal erosion phenomena. The same approach can be used to detect various types of hydromechanical disturbances at larger scales.

Description: We propose a two-phase damage theory in a viscoelastic medium to study the pressure and porosity diffusion in fractured near-surface porous rocks. The key ingredient in the viscoelastic theory is that the pressure difference between solid and fluid is divided into three parts, which contribute to reversible elastic potential energy, irreversible viscous entropy production and surface energy stored during deformation. The resulting continuum description of weakening and failure (distributed void generation and microcracking) in a linear Kelvin body accounts for surface energy being created by both viscous and elastic deformational work. The model shows that while non-linear permeability models leads to an enhanced diffusivity, damage makes the matrix more compressible if we assume the geometry/size of cracks remain unchanged. The net effect is that the porosity diffusivity is reduced causing fluid infiltration to accumulate closer to the injection source, leading to a slower fluid diffusion during hydraulic fracturing with a fixed porosity boundary condition. However if a constant overpressure boundary condition is applied, a weakened matrix with damage leads to greater pressure diffusivity than for porosity.

Description: We define an algorithm in the time domain for computing the unwrapped instantaneous phase and its derivative, the instantaneous frequency, using only derivatives and integrals. It does not require user-defined parameters, like most algorithms proposed so far. We validate and compare its performance with respect to open-source and commercial software by synthetic and real data examples.

Description: Simulating electromagnetic fields in the quasi-static regime by solving Maxwell's equations is a central task in many geophysical applications. In most cases, geophysical targets of interest exhibit complex topography and bathymetry as well as layers and faults. Capturing these effects with a sufficient level of detail is a huge challenge for numerical simulations. Standard techniques require a very fine discretization that can result in an impracticably large linear system to be solved. A remedy is to use locally refined and adaptive meshes, however, the potential coarsening is limited in the presence of highly heterogeneous and anisotropic conductivities. In this paper, we discuss the application of multiscale finite volume (MSFV) methods to Maxwell's equations in frequency domain. Given a partition of the fine mesh into a coarse mesh the idea is to obtain coarse-to-fine interpolation by solving local versions of Maxwell's equations on each coarsened grid cell. By construction, the interpolation accounts for fine scale conductivity changes, yields a natural homogenization, and reduces the fine mesh problem dramatically in size. To improve the accuracy for singular sources, we use an irregular coarsening strategy. We show that using MSFV methods we can simulate electromagnetic fields with reasonable accuracy in a fraction of the time as compared to state-of-the-art solvers for the fine mesh problem, especially when considering parallel platforms.

Description: A major challenge in metabolic engineering and synthetic biology is to balance the flux of an engineered heterologous metabolic pathway to achieve high yield and productivity in a target organism. Here, we report a simple, efficient and programmable approach named ‘customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER)’ for rapid tuning of gene expression in a heterologous pathway under distinct metabolic backgrounds. Specifically, a library of mutant pathways is created by de novo assembly of promoter mutants of varying strengths for each pathway gene in a target organism followed by high-throughput screening/selection. To demonstrate this approach, a single round of COMPACTER was used to generate both a xylose utilizing pathway with near-highest efficiency and a cellobiose utilizing pathway with highest efficiency that were ever reported in literature for both laboratory and industrial yeast strains. Interestingly, these engineered xylose and cellobiose utilizing pathways were all host-specific. Therefore, COMPACTER represents a powerful approach to tailor-make metabolic pathways for different strain backgrounds, which is difficult if not impossible to achieve by existing pathway engineering methods.

Description: We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.

Description: Wave-equation tomography (WT) and full waveform inversion (FWI) are combined through a hybrid misfit function to estimate high-resolution subsurface structures starting from a poorly constrained initial velocity model. Both methods share the same wavefield forward modelling and inversion schemes, while they differ only on the ways to calculate misfit functions and hence the ways to sample in the model space. Aiming at minimizing the cross-correlation phase delay between synthetic and real data, WT can be used to retrieve the long- and middle-wavelength model components, which are essential to FWI. Compared to ray-based traveltime tomography that is based on asymptotic high-frequency approximation, WT provides a better resolution by exploring the band-limited feature of seismic wavefield. On the other hand, FWI is capable of resolving the short-wavelength model component, complementing the WT. In this study, we apply WT to surface first-arrival refraction data, and apply FWI to both refraction and reflection data. We assign adaptive weights to the two different misfit measurements and build a progressive inversion strategy. To illustrate the advantage of our strategy over conventional ‘ray tomography + FWI’ approach, we show in a synthetic lens test that WT can provide extra subsurface information that is critical for a successful FWI application. To further show the efficiency, we test our strategy on the 2-D Marmousi model where satisfactory inversion results are achieved without much manual intervention. Finally, we apply the inversion strategy to a deep-water seismic data set acquired offshore Sumatra with a 12-km-long streamer. In order to alleviate several practical problems posed by the deep-water setting, we apply downward continuation (DC) to generate a virtual ocean bottom experiment data set prior to inversion. The new geometry after DC boosts up the shallow refractions, as well as avoiding cumbersome modelling through the thick water column, thus reducing the computation cost by 85 per cent. The inversion result from the new data set shows high-resolution shallow sediment structures and the migration images prove the superiority of the inverted model over a conventional tomography model.

Description: The activation of Late Quaternary faults in the Central Apennines (Italy) could generate earthquakes with magnitude of about 6.5, and the Monte Vettore fault system probably belongs to the same category of seismogenetic faults. Such structure has been defined ‘silent’, because of its geological and geomorphological evidences of past activation, but the absence of historical records in the seismic catalogues to be associated with its activation. The ‘Piano di Castelluccio’ intramountain basin, resulting from the Quaternary activity of normal faults, is characterized by a secondary fault strand highlighted by a NW–SE fault scarp: it has been already studied through palaeoseismological trenches, which highlighted evidences of Quaternary shallow faulting due to strong earthquakes, and through a 2-D ground penetrating radar (GPR) survey, showing the first geophysical signature of faulting for this site. Within the same place, a 3-D GPR volume over a 20 20 m area has been collected. The collection of radar echoes in three dimensions allows to map both the vertical and lateral continuity of shallow geometries of the fault zone (Fz), imaging features with high resolution, ranging from few metres to centimetres and therefore imaging also local variations at the microscale. Several geophysical markers of faulting, already highlighted on this site, have been taken as reference to plan the 3-D survey. In this paper, we provide the first 3-D subsurface imaging of an active shallow fault belonging to the Umbria-Marche Apennine highlighting the subsurface fault geometry and the stratigraphic sequence up to a depth of about 5 m. From our data, geophysical faulting signatures are clearly visible in three dimensions: diffraction hyperbolas, truncations of layers, local attenuated zones and varying dip of the layers have been detected within the Fz. The interpretation of the 3-D data set provided qualitative and quantitative geological information in addition to the fault location, like its geometry, boundaries and an estimation of the fault throw.

Description: To estimate the seismic hazard, the geometry (dip, length and orientation) and the dynamics (type of displacements and amplitude) of the faults in the area of interest need to be understood. In this paper, in addition to geomorphologic observations, we present the results of two ground penetrating radar (GPR) campaigns conducted in 2010 and 2011 along the Emeelt fault in the vicinity of Ulaanbaatar, capital of Mongolia, located in an intracontinental region with low deformation rate that induces long recurrence time between large earthquakes. As the geomorphology induced by the fault activity has been highly smoothed by erosion processes since the last event, the fault location and geometry is difficult to determine precisely. However, by using GPR first, a non-destructive and fast investigation, the fault and the sedimentary deposits near the surface can be characterized and the results can be used for the choice of trench location. GPR was performed with a 50 MHz antenna over 2-D lines and with a 500 MHz antenna for pseudo-3-D surveys. The 500 MHz GPR profiles show a good consistency with the trench observations, dug next to the pseudo-3-D surveys. The 3-D 500 MHz GPR imaging of a palaeochannel crossed by the fault allowed us to estimate its lateral displacement to be about 2 m. This is consistent with a right lateral strike-slip displacement induced by an earthquake around magnitude 7 or several around magnitude 6. The 2-D 50 MHz profiles, recorded perpendicular to the fault, show a strong reflection dipping to the NE, which corresponds to the fault plane. Those profiles provided complementary information on the fault such as its location at shallow depth, its dip angle (from 23° to 35°) and define its lateral extension.

Description: A new method to obtain the statistics of a geostatistical model is introduced. The method elicits the statistical information from a geological expert directly, by iteratively updating a population of vectors of statistics, based on the expert's subjective opinion of the corresponding geological simulations. Thus, it does not require the expert to have knowledge of the mathematical and statistical details of the model. The process uses a genetic algorithm to generate new vectors. We demonstrate the methodology for a particular geostatistical model used to model rock pore-space, which simulates the spatial distribution of matrix and pores over a 2-D grid, using multipoint statistics specified by conditional probabilities. Experts were asked to use the algorithm to estimate the statistics of a given target pore-space image with known statistics; thus, their numerical rates of convergence could be calculated. Convergence was measured for all experts, showing that the algorithm can be used to find appropriate probabilities given the expert's subjective input. However, considerable and apparently irreducible residual misfit was found between the true statistics and the estimates of statistics obtained by the experts, with the root-mean-square error on the conditional probabilities typically 〉0.1. This is interpreted as the limit of the experts’ abilities to distinguish between realizations of different spatial statistics using the algorithm. More accurate discrimination is therefore likely to require complementary elicitation techniques or sources of information independent of expert opinion.

Description: We show analytically that a well-known transfer function previously derived for the scalar acoustic problem that converts measurements from a 3-D (real-world) setting to a 2-D equivalent is directly applicable to the vector electromagnetic borehole ground penetrating radar problem. We also show that the transfer function's precision is improved for the low-loss case through the use of complex velocity. The transfer function has a strong effect on amplitude, and is therefore a critical preprocessing step for 2-D full-wave inversion when finding conductivity is of concern. We demonstrate the effectiveness of the transfer function through various numerical experiments and a synthetic frequency-domain full-wave inversion. We also compare the effectiveness of this curved-ray transfer function to a quasi-straight-ray transfer function. The inversion demonstrates the positive effect the transfer functions have on recovering conductivity and also that they are effective even when there are sharp velocity contrasts.

Description: We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.

Description: RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.

Description: Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for 〉100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo .

Description: Assembly of DNA ‘parts’ to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides—‘Clips’. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.

Description: We report and analyse the tsunami recorded in the northwestern Indian Ocean at the Makran region following the M w 7.7 Pakistan inland strike-slip earthquake on 2013 September 24. We analyse eleven tide gauge records as well as one DART record of this tsunami and perform numerical modelling of the tsunami that would be triggered by a range of possible sources. The tsunami registered a maximum wave height of 109 cm at the Qurayat tide gauge station (Oman). The dominant period of the tsunami was around 12 min, although wavelet analysis showed that parts of the tsunami energy were partitioned into a slightly wider period range of 7 and 16 min. Tsunami backward ray tracing showed that the tsunami source was possibly located offshore Jiwani (Pakistan) and that the tsunami was most likely triggered by the main shock. The aftershocks are distributed in the inland region and the coseismic vertical and horizontal displacements are also limited inland implying that the tsunami was generated by secondary sources triggered by the earthquake. Different possible tsunami sources including a mud volcano at the location of the newly generated island, and a mud volcano or diapir at offshore deep water were examined through numerical modelling and all failed to reproduce the observed waveforms. Numerical modelling showed that a submarine slump with a source dimension of about 10–15 km and a thickness of about 100 m located at 61.49°E and 24.62°N, that is, about 60–70 km off the Jiwani coast (Pakistan), seems capable of reasonably reproducing the wave amplitudes and periods of the observed tsunami waveforms. This event was the second instrumentally recorded tsunami in the region, after the Makran tsunami of 1945 November, and provides evidence for a hazard from landslide/slump-generated waves following seismic activity in the area.

Description: Joint inversion of different geophysical data sets is becoming a more popular and powerful tool, and it has been performed on data sensitive both to the same physical parameter and to different physical parameters. Joint inversion is undertaken to reduce acceptable model space and to increase sensitivity to model parameters that one method alone is unable to resolve adequately. We examine and implement a novel hybrid joint inversion approach. In our inversion scheme a model—the reference model—is fixed, and the information shared with the subsurface structure obtained from another method will be maximized; in our case conductivity structures from magnetotelluric (MT) inversion. During inversion, the joint probability distribution of the MT and the specified reference model is estimated and its entropy minimized in order to guide the inversion result towards a solution that is statistically compatible with the reference model. The powerful feature of this technique is that no explicit relationships between estimated model parameters and reference model ones are presumed: if a link exists in data then it is highlighted in the estimation of the joint probability distribution, if no link is required, then none is enforced. Tests performed verify the robustness of this method and the advantages of it in a 1-D anisotropic scenario are demonstrated. A case study was performed with data from Central Germany, effectively fitting an MT data set from a single station within as minimal an amount of anisotropy as required.

Description: Several slope failures are observed near the deformation front on the frontal ridges of the northern Cascadia accretionary margin off Vancouver Island. The cause for these events is not clear, although several lines of evidence indicate a possible connection between the occurrence of gas hydrate and submarine landslide features. The presence of gas hydrate is indicated by a prominent bottom-simulating reflector (BSR), at a depth of ~265–275 m beneath the seafloor (mbsf), as interpreted from vertical-incidence and wide-angle seismic data beneath the ridge crests of the frontal ridges. For one slide, informally called Slipstream Slide, the velocity structure inferred from tomography analyses shows anomalous high velocities values of about 2.0 km s –1 at shallow depths of 100 mbsf. The estimated depth of the glide plane (100 ± 10 m) closely matches the depth of these shallow high velocities. In contrast, at a frontal ridge slide just to the northwest (informally called Orca Slide), the glide plane occurs at the same depth as the current BSR. Our new results indicate that the glide plane of the Slipstream slope failure is associated with the contrast between sediments strengthened by gas hydrate and overlying sediments where little or no hydrate is present. In contrast, the glide plane of Orca Slide is between sediment strengthened by hydrate underlain by sediments beneath the gas hydrate stability zone, possibly containing free gas. Additionally, a set of margin perpendicular normal faults are imaged from seafloor down to BSR depth at both frontal ridges. As inferred from the multibeam bathymetry, the estimated volume of the material lost during the slope failure at Slipstream Slide is about 0.33 km 3 , and ~0.24 km 3 of this volume is present as debris material on the ocean basin floor. The 20 per cent difference is likely due to more widely distributed fine sediments not easily detectable as bathymetric anomalies. These volume estimates on the Cascadia margin are approaching the mass failure volume for other slides that have generated large tsunamis—for example 1–3 km 3 for a 1998 Papua New Guinea slide.

Description: The method presented here aims to assess the tsunami threat very rapidly after the occurrence of a large earthquake, using as input the parameters of the seismic source, and an approach based on Green's summation. We show that the main weakness of the approach (the need to consider only linear shallow water propagation) is largely compensated by the advantages in terms of computing performance and independence with respect to pre-computed scenarios. To test the approach and to illustrate its implementation in a real environment, we focus on the Sea of Oman, a tsunamigenic area characterized by Makran subduction zone which detailed structure is partially unknown and where secondary tsunami sources must also be taken into account, both for hazard studies and warning purposes. The potential source area is partitioned into a grid of unity water sources. A shallow water (SW) numerical model is used to pre-compute the corresponding empirical Green's functions on several points of interest located on the coasts of Iran, Pakistan and Oman. The comparison between Green's summation and the direct SW computation using the full resolution of the bathymetric grid shows that the accuracy is good enough for practical applications.

Description: Clay minerals as products of hydrothermal alteration significantly influence the hydraulic and mechanical properties of crystalline rock. Therefore, the localization and characterization of alteration zones by downhole measurements is a great challenge for the development of geothermal reservoirs. The magnetite bearing granite of the geothermal site in Soultz-sous-Forêts (France) experienced hydrothermal alteration during several tectonic events and clay mineral formation is especially observed in alteration halos around fracture zones. During the formation of clay minerals, magnetite was oxidized into hematite, which significantly reduces the magnetic susceptibility of the granite from ferrimagnetic to mostly paramagnetic values. The aim of this study was to find out if there exists a correlation between synthetic clay content logs (SCCLs) and measurements of magnetic susceptibility on cuttings in the granite in order to characterize their alteration mineralogy. Such a correlation has been proven for core samples of the EPS1 reference well. SCCLs were created from gamma ray and fracture density logs using a neural network. These logs can localize altered fracture zones in the GPK1-4 wells, where no core material is available. Mass susceptibility from 261 cutting samples of the wells GPK1–GPK4 was compared with the neural network derived synthetic logs. We applied a combination of temperature dependent magnetic susceptibility measurements with optical and electron microscopy, and energy dispersive X-ray spectroscopy to discriminate different stages of alteration. We found, that also in the granite cuttings an increasing alteration grade is characterized by an advancing oxidation of magnetite into hematite and a reduction of magnetic susceptibility. A challenge to face for the interpretation of magnetic susceptibility data from cuttings material is that extreme alteration grades can also display increased susceptibilities due to the formation of secondary magnetite. Low magnetic susceptibility can also be attributed to primary low magnetite content, if the granite facies changes. In order to interpret magnetic susceptibility from cuttings, contaminations with iron from wear debris of the drilling tools must be eliminated. Provided that the magnetic mineralogy of the granite is known in detail, this method in combination with petrographic investigations is suited to indicate and characterize hydrothermal alteration and the appearance of clay.

Description: Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni , which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.

Description: Elastic and mechanical weakening from water saturation are widely known to occur in sedimentary rocks, and particularly in carbonate rocks. To improve our understanding of the physics underlying this phenomenon, ultrasonic ( f  ~ 0.5 MHz) elastic properties are measured on a large suite of clean limestones and sandstones at very low saturations from relative humidity (RH) variations at ambient conditions. Measurements clearly highlight an elastic weakening (i.e. decrease in elastic wave velocity) from moisture adsorption. P - and S -wave velocities are similarly affected by adsorption, but in a different way for limestones and sandstone samples. While the elastic properties of limestone samples show almost no RH dependence, a large weakening is observed for samples of Fontainebleau sandstone that increases with the samples’ porosity. The main elastic weakening effect is likely to result from adsorption of fluid at grain contacts. It thus affects particularly granular rocks such as sandstones while well-cemented limestones are not affected. The granular model from Murphy et al. , accounting for surface energy effects, proves to be appropriate. Applying this model, it is shown that (i) P - and S -wave velocities have the same dependence on surface energy, which is consistent with the measurements and (ii) surface energy values obtained from the ultrasonic data using this model correlate with RH, and are consistent with the expected value for quartz crystals at vapour pressure. Yet, porosity, which relates to degree of cementation in the particular case of Fontainebleau sandstone, appears to be an additional parameter. A modified model is thus derived using the cementation model from Digby, accounting for a bonding radius at grain contact. It proves to apply well to the measured data. The fundamental difference between limestones’ and sandstones’ dependence to RH appears to be related to a microstructural difference. Saturation variations from RH increase depend on specific surface area, which is particularly low in Fontainebleau sandstones and large in microporous limestones. However elastic weakening from RH is more important in sandstones owing to their granular microstructure.

Description: A novel experimental method is introduced to estimate the Thomsen's elastic anisotropy parameters and of a transversely isotropic shale under variable stress and saturation conditions. The method consists in recording P -wave velocities along numerous paths on a cylindrical specimen using miniature ultrasonic transducers. Such an overdetermined set of measurements is specifically designed to reduce the uncertainty associated with the determination of Thomsen's parameter compared to the classical method for which a single off-axis measurement is used (usually at 45° to the specimen's axis). This method is applied to a specimen of Opalinus Clay recovered from the Mont-Terri Underground Research Laboratory in Switzerland. The specimen is first saturated with brine at low effective pressure and then subjected to an effective pressure cycle up to 40 MPa, followed by a triaxial loading up to failure. During saturation and deformation, the evolution of P -wave velocities along a maximum of 240 ray paths is monitored and Thomsen's parameters α , and are computed by fitting Thomsen's weak anisotropy model to the data. The values of and obtained at the highest confining pressures reached during the experiment are comparable with those predicted from X-ray diffraction texture analysis and modelling for Opalinus Clay reported in the literature. These models neglect the effect of soft-porosity on elastic properties, but become relevant when soft porosity is closed at high effective pressure.

Description: We present a novel accurate and efficient goal-oriented adaptive finite-element method solution for complex multi-electrodes resistivity system with arbitrary smooth surface topographies. A simple Green's function of a half-space model is adopted to eliminate the singularity. A unified boundary value problem for the regular potential is formulated for a multi-electrodes system so that it shares a common system matrix. In addition, a goal-oriented error estimation technique is developed to generate an optimal common grid so that highly accurate solutions are obtained with minimum computation cost. Synthetic models are used to verify our algorithm and excellent agreements are obtained by comparing with other methods.

Description: Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K d ) 〈10 pM, representing a ~200-fold improvement in binding affinity over the monomeric aptamers and a ~15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

Description: Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), -butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and -butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapies.

Description: The Geological Survey of Sweden has been collecting airborne tensor very low frequency data (VLF) over several decades, covering large parts of the country. The data has been an invaluable source of information for identifying conductive structures that can among other things be related to water-filled fault zones, wet sediments that fill valleys or ore mineralizations. Because the method only uses two differently polarized plane waves of very similar frequency, vertical resolution is low and interpretation is in most cases limited to maps that are directly derived from the data. Occasionally, 2-D inversion is carried out along selected profiles. In this paper, we present for the first time a 3-D inversion for tensor VLF data in order to further increase the usefulness of the data set. The inversion is performed using a non-linear conjugate gradient scheme (Polak-Ribière) with an inexact line-search. The gradient is obtained by an algebraic adjoint method that requires one additional forward calculation involving the adjoint system matrix. The forward modelling is based on integral equations with an analytic formulation of the half-space Green's tensor. It avoids typically required Hankel transforms and is particularly amenable to singularity removal prior to the numerical integration over the volume elements. The system is solved iteratively, thus avoiding construction and storage of the dense system matrix. By using fast 3-D Fourier transforms on nested grids, subsequently farther away interactions are represented with less detail and therefore with less computational effort, enabling us to bridge the gap between the relatively short wavelengths of the fields (tens of metres) and the large model dimensions (several square kilometres). We find that the approximation of the fields can be off by several per cent, yet the transfer functions in the air are practically unaffected. We verify our code using synthetic calculations from well-established 2-D methods, and trade modelling accuracy off against computational effort in order to keep the inversion feasible in both respects. Our compromise is to limit the permissible resistivity to not fall below 100 m to maintain computational domains as large as 10 10 km 2 and computation times on the order of a few hours on standard PCs. We investigate the effect of possible local violations of these limits. Even though the conductivity magnitude can then not be recovered correctly, we do not observe any structural artefacts related to this in our tests. We invert a data set from northern Sweden, where we find an excellent agreement of known geological features, such as contacts or fault zones, with elongated conductive structures, while high resistivity is encountered in probably less disturbed geology, often related to topographic highs, which have survived predominantly glacial erosion processes. As expected from synthetic studies, the resolution is laterally high, but vertically limited down to the top of conductive structures.

Description: Induced polarization is a geophysical method looking to image and interpret low-frequency polarization mechanisms occurring in porous media. Below 10 kHz, the quadrature conductivity of metal-free sandy and clayey materials exhibits a distribution of relaxation times, which can be related to the pore size distribution of these porous materials. When the polarization spectra are fitted with a Cole–Cole model, we first observe that the main relaxation time is controlled by the main pore size of the material and that the Cole–Cole exponent c is never much above 0.5, a value corresponding to a Warburg function. The complex conductivity is then obtained through a convolution product between the pore size distribution and such Warburg function. We also provide a way to recover the pore size distribution by performing a deconvolution of measured spectra using the Warburg function. A new dataset of mercury porosimetry and induced polarization data of six siliciclastic materials supports the hypothesis that the Cole–Cole relaxation time is strongly controlled by the pore size, and especially the characteristic pore size corresponding to the peak of the pore size distribution from mercury porosimetry. The distribution of the pore throat sizes of these materials seems fairly well recovered using the Warburg decomposition of the spectral induced polarization spectra but additional data will be needed to confirm this finding.

Description: We propose a new, simple and efficient method to image electrical resistivity between a set of wells. Our procedure consists of two steps: first, we map the interfaces between various subsurface formations using seismoelectric conversions; second, we derive the formation resistivity using image-guided cross-well electric tomography. In the first step, we focus seismic energy at a set of points located on a regular grid between wells, which enables us to map the geological formations in terms of heterogeneities in electrical, hydraulic and/or seismic properties. The density of the scanning points (i.e. the seismoelectric image resolution) is related to the wavelength of the seismic impulse used to scan the formations. Each time the seismic energy is focused at a point, the resulting electrical potential burst (equivalent to the one generated by a volumetric seismic source) is recorded remotely at a set of electrodes positioned in wells (the reference electrode can be located on the ground surface or far enough to be considered at infinity). We construct a high-resolution ‘seismoelectric’ image by assigning the electrical potential simulated at these fixed electrodes to the location of the seismic focus. In a follow-up step, the structure of this image is used in image-guided inversion to improve electrical resistivity tomography between the two wells. The structural information from the seismoelectric image is used to impose constraints on the model covariance matrix used in the inversion of the electrical resistivity data. This approach offers new perspectives in recovering fine structure of resistivity (high definition resistivity tomography) between the wells, which cannot be resolved through conventional cross-well resistivity or from seismic tomography alone.

Description: In subduction zones, shallow subsurface structures are the manifestation of the plate interactions at depth. However, significant water depths, rough bathymetry and presence of heavily deformed accretionary wedge materials hamper imaging of the near-surface features to a great extent using conventional imaging techniques. In this study, we show results using an integrated processing technique to a multichannel seismic data set acquired in 2006 from the northwestern offshore Sumatra. We start with first downward continuing the 12-km-long surface streamer data to the seafloor, followed by a high-resolution traveltime tomography of refracted phases to determine a detailed velocity–depth model of subsurface, which in turns, is used for pre-stack depth migration in order to delineate the shallow subsurface structures beneath the trench, subduction front and outer accretionary wedge. Our velocity–depth model and the depth migrated image depict variation of sediment properties across the front and structures of uppermost sedimentary sequence with an unprecedented high resolution providing the precise location of the frontal and conjugate thrusts, highly folded sedimentary sequences, which in turns describe their relationship with the top of the subducting plate and factors that control rupture propagation to the trench. Furthermore, we estimate the porosity distribution across the front, where we find a 12 and 18 per cent decrease in porosity beneath the deformation front and the inner accretionary plateau at 500 m below the seafloor, respectively, which we interpret to be due to the compaction. A significant decrease in porosity at the plate interface below 5–6-km thick sediments near the deformation front would increase the coupling, leading to the rupture propagation up to the trench, uplifting 4.5 km water and producing large tsunami.

Description: We present a scale- and parameter-adaptive method to pre-condition the gradient of the parameters to be inverted in time-domain 2-D elastic full-waveform inversion (FWI). The proposed technique, which relies on a change of variables of the model parameters, allows to balance the value of the gradient of the Lamé parameters and density throughout the model in each step of the multiscale inversion. The main difference compared to existing gradient pre-conditioners is that the variables are automatically selected based on a least-squares minimization criteria of the gradient weight, which corresponds to the product of the gradient by a power of the parameter to be inverted. Based on numerical tests made with (1) a modified version of the Marmousi-2 model, and (2) a high-velocity and density local anomaly model, we illustrate that the value of the power helps to balance the gradient throughout the model. In addition, we show that a particular value exists for each parameter that optimizes the inversion results in terms of accuracy and efficiency. For the two models, the optimal power is ~2.0–2.5 and ~1.5 for the first and second Lamé parameters, respectively; and between 3 and 6, depending on the inverted frequency, for density. These power values provide the fastest and most accurate inversion results for the three parameters in the framework of multiscale and multishooting FWI using three different optimization schemes.

Description: Cy3B is an extremely bright and stable fluorescent dye, which is only available for coupling to nucleic acids post-synthetically. This severely limits its use in the fields of genomics, biology and nanotechnology. We have optimized the synthesis of Cy3B, and for the first time produced a diverse range of Cy3B monomers for use in solid-phase oligonucleotide synthesis. This molecular toolkit includes phosphoramidite monomers with Cy3B linked to deoxyribose, to the 5-position of thymine, and to a hexynyl linker, in addition to an oligonucleotide synthesis resin in which Cy3B is linked to deoxyribose. These monomers have been used to incorporate single and multiple Cy3B units into oligonucleotides internally and at both termini. Cy3B Taqman probes, Scorpions and HyBeacons have been synthesized and used successfully in mutation detection, and a dual Cy3B Molecular Beacon was synthesized and found to be superior to the corresponding Cy3B/DABCYL Beacon. Attachment of Cy3, Cy3B and Cy5 to the 5-position of thymidine by an ethynyl linker enabled the synthesis of an oligonucleotide FRET system. The rigid linker between the dye and nucleobase minimizes dye–dye and dye–DNA interactions and reduces fluorescence quenching. These reagents open up new future applications of Cy3B, including more sensitive single-molecule and cell-imaging studies.

Description: Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices.

Description: Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 10 6 variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks.

Description: The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

Description: Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli , we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 x 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

Description: Studying complex biological processes such as cancer development, stem cell induction and transdifferentiation requires the modulation of multiple genes or pathways at one time in a single cell. Herein, we describe straightforward methods for rapid and efficient assembly of bacterial marker free multigene cassettes containing up to six complementary DNAs/short hairpin RNAs. We have termed this method RecWay assembly, as it makes use of both Cre recombinase and the commercially available Gateway cloning system. Further, because RecWay assembly uses truly modular components, it allows for the generation of randomly assembled multigene vector libraries. These multigene vectors are integratable, and later excisable, using the highly efficient piggyBac ( PB ) DNA transposon system. Moreover, we have dramatically improved the expression of stably integrated multigene vectors by incorporation of insulator elements to prevent promoter interference seen with multigene vectors. We demonstrate that insulated multigene PB transposons can stably integrate and faithfully express up to five fluorescent proteins and the puromycin-thymidine kinase resistance gene in vitro , with up to 70-fold higher gene expression compared with analogous uninsulated vectors . RecWay assembly of multigene transposon vectors allows for widely applicable modelling of highly complex biological processes and can be easily performed by other research laboratories.

Description: Techniques for assembly of designed DNA sequences are important for synthetic biology. So far, a few methods have been developed towards high-throughput seamless DNA assembly in vitro , including both the homologous sequences-based system and the type IIS-mediated system. Here, we describe a novel method designated ‘MASTER Ligation’, by which multiple DNA sequences can be seamlessly assembled through a simple and sequence-independent hierarchical procedure. The key restriction endonuclease used, MspJI, shares both type IIM and type IIS properties; thus, it only recognizes the methylation-specific 4-bp sites, m CNNR (R = A or G), and cuts DNA outside of the recognition sequences. This method was tested via successful assembly of either multiple polymerase chain reaction amplicons or restriction fragments of the actinorhodin biosynthetic cluster of Streptomyces coelicolor (~29 kb), which was further heterologously expressed in a fast-growing and moderately thermophilic strain, Streptomyces sp. 4F.

Description: Bacterial operons are nature’s tool for regulating and coordinating multi-gene expression in prokaryotes. They are also a gene architecture commonly used in the biosynthesis of many pharmaceutically important compounds and industrially useful chemicals. Despite being an important eukaryotic production host, Saccharomyces cerevisiae has never had such gene architecture. Here, we report the development of a system to assemble and regulate a multi-gene pathway in S. cerevisiae . Full pathways can be constructed using pre-made parts from a plasmid toolbox. Subsequently, through the use of a yeast strain containing a stably integrated gene switch, the assembled pathway can be regulated using a readily available and inexpensive compound—estradiol—with extremely high sensitivity (10 nM). To demonstrate the use of the system, we assembled the five-gene zeaxanthin biosynthetic pathway in a single step and showed the ligand-dependent coordinated expression of all five genes as well as the tightly regulated production of zeaxanthin. Compared with a previously reported constitutive zeaxanthin pathway, our inducible pathway was shown to have 50-fold higher production level.

Description: Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger–finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy—finger stitching—that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger–finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo , demonstrating that stitching yields ZFAs with robust recognition properties.

Description: The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ~800-fold dynamic range within Escherichia coli . We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies ( r = 0.9, n = 31) better than models trained on all terminators ( r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.

Description: Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate l -lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l -lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different l -lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum , the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.

Description: The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11 , were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

Description: Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.

Description: The generation of genome-modified animals is a powerful approach to analyze gene functions. The CAS9/guide RNA (gRNA) system is expected to become widely used for the efficient generation of genome-modified animals, but detailed studies on optimum conditions and availability are limited. In the present study, we attempted to generate large-scale genome-modified mice with an optimized CAS9/gRNA system, and confirmed the transmission of these mutations to the next generations. A comparison of different types of gRNA indicated that the target loci of almost all pups were modified successfully by the use of long-type gRNAs with CAS9. We showed that this system has much higher mutation efficiency and much lower off-target effect compared to zinc-finger nuclease. We propose that most of these off-target effects can be avoided by the careful control of CAS9 mRNA concentration and that the genome-modification efficiency depends rather on the gRNA concentration. Under optimized conditions, large-scale (~10 kb) genome-modified mice can be efficiently generated by modifying two loci on a single chromosome using two gRNAs at once in mouse zygotes. In addition, the normal transmission of these CAS9/gRNA-induced mutations to the next generation was confirmed. These results indicate that CAS9/gRNA system can become a highly effective tool for the generation of genome-modified animals.

Description: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.

Description: Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.