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: We present high-resolution tomographic images in source areas of 26 large crustal earthquakes ( M 6.0–7.2) which occurred in Northeast Japan (Tohoku) during the past 120 yr from 1894 to 2014. Prominent low-velocity (low- V ) and high Poisson's ratio (high- ) anomalies are revealed in the crust and mantle wedge under the source areas. Beneath the volcanic front and backarc areas, the low- V and high- zones reflect arc-magma related high-temperature anomalies which are produced by joint effects of corner flow in the mantle wedge and fluids from dehydration of the subducting Pacific slab. The hot anomalies cause locally thinning and weakening of the brittle seismogenic layer above them. Low-frequency micro-earthquakes are observed in the lower crust and uppermost mantle in or around the low- V zones, which reflect ascending of arc magma and fluids from the mantle wedge to the crust. No volcano and magma exist in the forearc area due to low temperature there, hence the low- V zones in the forearc reflect fluids from the slab dehydration. The ascending fluids may have produced a ‘water wall’ in the mantle wedge and crust beneath the forearc area. When the water enters active faults in the crust, the fault-zone friction is reduced and so large earthquakes can be induced. These results indicate that the nucleation of a large earthquake is not entirely a mechanical process, but is closely associated with subduction dynamics and physical and chemical properties of rocks in the crust and upper mantle. In particular, arc magma and fluids play an important role in the seismogenesis.

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: For a period of about 1 yr between the summers of 2010 and 2011, 25 broad-band seismographs were deployed in a roughly linear array across the eastern end of the Qaidam basin and the Qilian Shan in the northeastern Tibetan plateau. This region is probably the most suitable place to study the ongoing convergence interaction between the high Tibetan plateau and the main Asian continental plate. Low-frequency P receiver function analysis of the data provides an image of the crust and mantle down to 700 km depth. In addition to the Moho at 45–65 km depth beneath the profile, the 410 and 660 km discontinuities bounding the mantle transition zone can be identified at 400–410 and 650–660 km depths, respectively. A possible increase in temperature in the upper mantle thought to exist beneath the northern part of the high Tibetan plateau is thus confined to this part of the plateau and lower upper-mantle temperatures similar to those beneath southern Tibet occur beneath the Qaidam basin and Qilian Shan. When higher frequencies are included in the P receiver function analysis, a positive Ps converter dipping down to the south from 70–75 km depth at 37.9°N to about 110 km depth at 36°N is imaged. As this feature is only seen in high-frequency images and not in the low-frequency image, it is modelled as the positive Ps conversion from the base of an approximately 5-km-thick anisotropic layer at the top of the Asian mantle lithosphere which is currently subducting. This south-dipping converter continues to the south on the INDEPTH IV profile. S receiver function analysis completes the image of the structure below the Qilian Shan profile with the identification of the lithosphere–asthenosphere boundary (LAB). The LAB of the Asian Plate is identified for a reference slowness of 6.4 s deg –1 at 12–14 s (105–125 km depth) between 38 and 41°N below the northern part of the S receiver function profile. To the south it increases in depth such that it is at about 19 s (170 km depth) between 34 and 35°N at the southern end of the profile. The LAB of the Asian Plate occurs at similar depths on the INDEPTH IV profile at the latitudes where the INDEPTH IV and Qilian Shan profiles overlap. As on the INDEPTH IV profile to the south, between 34 and 35°N at the southern end of the Qilian Shan profile there is evidence from the S receiver functions for the LAB of a separate Tibetan Plate.

Description: Lower and upper bounds for present deformation rates across faults in central California between the San Andreas Fault and Pacific coast are estimated from a new Global Positioning System (GPS) velocity field for central, western California in light of geodetic evidence presented in a companion paper for slow, but significant deformation within the Pacific Plate between young seafloor in the eastern Pacific and older seafloor elsewhere on the plate. Transects of the GPS velocity field across the San Andreas Fault between Parkfield and San Juan Buatista, where fault slip is dominated by creep and the velocity field thus reveals the off-fault deformation, show that GPS sites in westernmost California move approximately parallel to the fault at an average rate of 3.4 ± 0.4 mm yr –1 relative to the older interior of the Pacific Plate, but only 1.8 ± 0.6 mm yr –1 if the Pacific Plate frame of reference is corrected for deformation within the plate. Modelled interseismic elastic deformation from the weakly coupled creeping segment of the San Andreas Fault is an order-of-magnitude too small to explain the southeastward motions of coastal sites in western California. Similarly, models that maximize residual viscoelastic deformation from the 1857 Fort Tejon and 1906 San Francisco earthquakes mismatch both the rates and directions of GPS site motions in central California relative to the Pacific Plate. Neither thus explains the site motions southwest of the San Andreas fault, indicating that the site motions measure deformation across faults and folds outboard of the San Andreas Fault. The non-zero site velocities thus constitute strong evidence for active folding and faulting outboard from the creeping segment of the San Andreas Fault and suggest limits of 0–2 mm yr –1 for the Rinconada Fault slip rate and 1.8 ± 0.6 to 3.4 ± 0.4 mm yr –1 for the slip rates integrated across near-coastal faults such as the Hosgri, San Gregorio and San Simeon faults.

Description: We combine new, well-determined GPS velocities from Clarion, Guadalupe and Socorro islands on young seafloor in the eastern Pacific basin with newly estimated velocities for 26 GPS sites from older seafloor in the central, western and southern parts of the Pacific Plate to test for deformation within the interior of the Pacific Plate and estimate the viscosity of the asthenosphere below the plate. Relative to a Pacific Plate reference frame defined from the velocities of the 26 GPS sites in other areas of the Pacific Plate, GPS sites on Clarion and Guadalupe islands in the eastern Pacific move 1.2 ± 0.6 mm yr –1 (1) towards S09°W ± 38° and 1.9 ± 0.3 mm yr –1 towards S19°E ± 10°, respectively. The two velocities, which are consistent within their 95 per cent uncertainties, both differ significantly from Pacific Plate motion. Transient volcanic deformation related to a 1993–1996 eruption of the Socorro Island shield volcano renders our GPS velocity from that island unreliable for the tectonic analysis although its motion is also southward like those of Clarion and Guadalupe islands. We test but reject the possibilities that drift of Earth's origin in ITRF2008 or unmodelled elastic offsets due to large-magnitude earthquakes around the Pacific rim since 1993 can be invoked to explain the apparent slow southward motions of Clarion and Guadalupe islands. Similarly, corrections to the Pacific Plate GPS velocity field for possible viscoelastic deformation triggered by large-magnitude earthquakes since 1950 also fail to explain the southward motions of the two islands. Viscoelastic models with prescribed asthenospheric viscosities lower than 1  x 10 19 Pa s instead introduce statistically significant inconsistencies into the Pacific Plate velocity field, suggesting that the viscosity of the asthenosphere below the plate is higher than 1  x 10 19 Pa s. Elastic deformation from locked Pacific–North America Plate boundary faults is also too small to explain the southward motions of the two islands. Horizontal thermal contraction of the plate interior may explain the motion observed at Clarion and Guadalupe islands, as might long-term tectonic deformation of the plate interior.

Description: In this study, a new method for computing the sensitivity of the glacial isostatic adjustment (GIA) forward solution with respect to the Earth's mantle viscosity, the so-called the forward sensitivity method (FSM), and a method for computing the gradient of data misfit with respect to viscosity parameters, the so-called adjoint-state method (ASM), are presented. These advanced formal methods complement each other in the inverse modelling of GIA-related observations. When solving this inverse problem, the first step is to calculate the forward sensitivities by the FSM and use them to fix the model parameters that do not affect the forward model solution, as well as identifying and removing redundant parts of the inferred viscosity structure. Once the viscosity model is optimized in view of the forward sensitivities, the minimization of the data misfit with respect to the viscosity parameters can be carried out by a gradient technique which makes use of the ASM. The aim is this paper is to derive the FSM and ASM in the forms that are closely associated with the forward solver of GIA developed by Martinec. Since this method is based on a continuous form of the forward model equations, which are then discretized by spectral and finite elements, we first derive the continuous forms of the FSM and ASM and then discretize them by the spectral and finite elements used in the discretization of the forward model equations. The advantage of this approach is that all three methods (forward, FSM and ASM) have the same matrix of equations and use the same methodology for the implementation of the time evolution of stresses. The only difference between the forward method and the FSM and ASM is that the different numerical differencing schemes for the time evolution of the Maxwell and generalized Maxwell viscous stresses are applied in the respective methods. However, it requires only a little extra computational time for carrying out the FSM and ASM numerically. An straightforward approach to compute the gradient of the data misfit is the brute-force method, whereby the partial derivatives of the misfit with respect to model parameters are approximated by the centred difference of two forward model runs. Although the brute-force method is useful for computing the gradient of the data misfit with respect to a small number of model parameters, it becomes expensive for a viscosity model with a large number of parameters. The ASM offers an efficient alternative for computing the gradient of the misfit since the computational time of the ASM is independent of the number of viscosity parameters. The ASM is thus highly efficient for calculating the gradient of the misfit for models with large numbers of parameters. However, the forward-model solution for each time step must be stored, hence the memory demands scale linearly with the number of time steps. This is the main drawback of the ASM.

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: Relative to the gravitational potential energy of the Earth's monopole, the multipole energy has received far less attention. In this paper, we recapitulate the basic physics from first principles and derive the formulas for multipole energies in analogy to classical electrostatic theory. We focus on the zonal quadrupole energy associated with the Earth's oblateness, the dominant term in Earth's gravity field apart from the monopole. We find the gravitational energy E oblateness 10 –6 | E monopole | = +2.5 x 10 26 J. We examine the implications of E oblateness and its changes associated with long-term ‘secular’ decreases in the oblateness parameter J 2 . We find the rate of loss of E oblateness due to the Earth rounding induced by the present-day GIA is about –200 GW, an amount quite significant in the kinetic energy budget of the mantle heat engine that drives the plate tectonics that has been estimated to be ~1 TW. We also assert that the tidal braking and the global earthquake dislocations, both resulting in Earth rounding on long-term geological timescales, are accompanied with a secular decrease of E oblateness at nearly the same rate of several GW.

Description: Large-scale chemical lateral heterogeneities are inferred in the Earth's lowermost mantle by seismological studies. We explore the model space of thermochemical convection that can maintain reservoirs of dense material for a long period of time, by using similar analysis in 3-D spherical geometry. In this study, we focus on the parameters thought to be important in controlling the stability and structure of primordial dense reservoirs in the lower mantle, including the chemical density contrast between the primordial dense material and the regular mantle material (buoyancy ratio), thermal and chemical viscosity contrasts, volume fraction of primordial dense material and the Clapeyron slope of the phase transition at 660 km depth. We find that most of the findings from the 3-D Cartesian study still apply to 3-D spherical cases after slight modifications. Varying buoyancy ratio leads to different flow patterns, from rapid upwelling to stable layering; and large thermal viscosity contrasts are required to generate long wavelength chemical structures in the lower mantle. Chemical viscosity contrasts in a reasonable range have a second-order role in modifying the stability of the dense anomalies. The volume fraction of the initial primordial dense material does not effect the results with large thermal viscosity contrasts, but has significant effects on calculations with intermediate and small thermal viscosity contrasts. The volume fraction of dense material at which the flow pattern changes from unstable to stable depends on buoyancy ratio and thermal viscosity contrast. An endothermic phase transition at 660 km depth acts as a ‘filter’ allowing cold slabs to penetrate while blocking most of the dense material from penetrating to the upper mantle.

Description: Relative sea level curves contain coupled information about absolute sea level change and vertical lithospheric movement. Such curves may be constructed based on, for example tide gauge data for the most recent times and different types of geological data for ancient times. Correct account for vertical lithospheric movement is essential for estimation of reliable values of absolute sea level change from relative sea level data and vise versa. For modern times, estimates of vertical lithospheric movement may be constrained by data (e.g. GPS-based measurements), which are independent from the relative sea level data. Similar independent data do not exist for ancient times. The purpose of this study is to test two simple inversion approaches for simultaneous estimation of lithospheric uplift rates and absolute sea level change rates for ancient times in areas where a dense coverage of relative sea level data exists and well-constrained average lithospheric movement values are known from, for example glacial isostatic adjustment (GIA) models. The inversion approaches are tested and used for simultaneous estimation of lithospheric uplift rates and absolute sea level change rates in southwest Scandinavia from modern relative sea level data series that cover the period from 1900 to 2000. In both approaches, a priori information is required to solve the inverse problem. A priori information about the average vertical lithospheric movement in the area of interest is critical for the quality of the obtained results. The two tested inversion schemes result in estimated absolute sea level rise of ~1.2/1.3 mm yr –1 and vertical uplift rates ranging from approximately –1.4/–1.2 mm yr –1 (subsidence) to about 5.0/5.2 mm yr –1 if an a priori value of 1 mm yr –1 is used for the vertical lithospheric movement throughout the study area. In case the studied time interval is broken into two time intervals (before and after 1970), absolute sea level rise values of ~0.8/1.2 mm yr –1 (before 1970) and ~2.0 mm yr –1 (after 1970) are found. The uplift patterns resulting from the different inversions suggest that the lithospheric post-GIA response changes near the border between the Danish Basin and the Fennoscandian Shield. The obtained patterns of vertical lithospheric movement rates are comparable to results from other studies based on different and similar data types. Main differences between the inversion results and the results from other studies are caused by factors such as the simplifications included in the inversion approach, such as neglecting local sea level variation caused by the dominant wind patterns, and the a priori values chosen for the vertical uplift rates. The tests of the inversion schemes reveal that realistic values of absolute sea level rise and lithospheric uplift may be simultaneously estimated provided that reliable prior knowledge regarding the overall lithospheric uplift in the study area is available beforehand. In the presented parametrizations, only one absolute sea level change rate value is estimated for each studied time interval while several vertical movement rates are found, and the inverse estimate of absolute sea level change rate is practically insensitive with respect to the choice of a priori value of absolute sea level change, as long as the uncertainty assigned to this a priori value is kept sufficiently high.

Description: In 1356, a magnitude 6–7 earthquake occurred near Basel, in Switzerland. But recent compilations of GPS measurements reveal that measured horizontal deformation rates in northwestern continental Europe are smaller than error bars on the measurements, proving present tectonic activity, if any, is very small in this area. We propose to reconcile these apparently antinomic observations with a mechanical model of the lithosphere that takes into account the geometry of the lithosphere–asthenosphere boundary, assuming that the only loading mechanism is gravity. The lithosphere is considered to be an elastoplastic material satisfying a Von Mises plasticity criterion. The model, which is 400 km long, 360 km wide and 230 km thick, is centred near Belfort in eastern France, with its width oriented parallel to the N145°E direction. It also takes into account the real topography of both the ground surface and that of the Moho discontinuity. Not only does the model reproduce observed principal stress directions orientations, it also identifies a plastic zone that fits roughly the most seismically active domain of the region. Interestingly, a somewhat similar stress map may be produced by considering an elastic lithosphere and an ad-hoc horizontal ‘tectonic’ stress field. However, for the latter model, examination of the plasticity criterion suggests that plastic deformation should have taken place. It is concluded that the present-day stress field in this region is likely controlled by gravity and rheology, rather than by active Alpine tectonics.

Description: We have investigated variations in transition zone thickness under the Borborema Province of NE Brazil by migrating and stacking teleseismic P -wave receiver functions at 32 seismic stations in the region. The Borborema Province represents the western portion of a larger Neoproterozoic mobile belt that occupied much of northern Gondwana, where extensional processes in the Mesozoic lead to the formation of a number of intracontinental basins and ultimately continental breakup. Episodes of intraplate volcanism and uplift marked the evolution of the Province during the Cenozoic, but it is unclear whether those episodes originated from shallow or deep-seated magmatic sources. On one hand, the elliptical shape of the uplifted area, the stress pattern of the Cenozoic deformation and the time overlap between uplift and volcanism suggest doming from thermal activation due to a deep-seated mantle plume. On the other hand, geochronological dates of volcanic bodies in the Province are better understood if resulting from lithospheric erosion by a shallow, small-scale convection cell. Large temperature anomalies are expected to be associated with mantle upwellings, and constraints on the depth extent of the upwellings can be obtained from transition zone thickness. Thinning of the transition zone with respect to its nominal 250 km value is considered diagnostic for positive temperature anomalies, while thickening is considered diagnostic for negative anomalies. Our results show that transition zone thickness is normal, around 250 km, throughout the Province and suggest that thermal perturbations—if present—are confined to the upper mantle. We argue that our results are consistent with a local, shallow magmatic source for the Cenozoic intraplate volcanism of the Borborema Province, although other proposed scenarios—such as channeling of upwelling plume material along lithospheric thin spots—cannot be ruled out with our analysis.

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: Standard techniques for computed tomography imaging are not directly applicable to a carbonate rock because of the geometric complexity of its pore space. In this study, we first characterized the pore structure in Majella limestone with 30 per cent porosity. Microtomography data acquired on this rock was partitioned into three distinct domains: macropores, solid grains, and an intermediate domain made up of voxels of solid embedded with micropores below the resolution. A morphological analysis of the microtomography images shows that in Majella limestone both the solid and intermediate domains are interconnected in a manner similar to that reported previously in a less porous limestone. We however show that the macroporosity in Majella limestone is fundamentally different, in that it has a percolative backbone which may contribute significantly to its permeability. We then applied for the first time 3-D-volumetric digital image correlation (DIC) to characterize the mode of mechanical failure in this limestone. Samples were triaxially deformed over a wide range of confining pressures. Tomography imaging was performed on these samples before and after deformation. Inelastic compaction was observed at all tested pressures associated with both brittle and ductile behaviors. Our DIC analysis reveals the structure of compacting shear bands in Majella limestone deformed in the transitional regime. It also indicates an increase of geometric complexity with increasing confinement—from a planar shear band, to a curvilinear band, and ultimately to a diffuse multiplicity of bands, before shear localization is inhibited as the failure mode completes the transition to delocalized cataclastic flow.

Description: Wide-angle reflection/refraction seismic profiles were recorded across the Cyprus Arc, the plate boundary between the African Plate and the Aegean–Anatolian microplate, from the Eratosthenes Seamount to the Hecataeus Rise immediately south of Cyprus. The resultant models were able to resolve detail of significant lateral velocity variations, though the deepest crust and Moho are not well resolved from the seismic data alone. Conclusions from the modelling suggest that (i) Eratosthenes Seamount consists of continental crust but exhibits a laterally variable velocity structure with a thicker middle crust and thinner lower crust to the northeast; (ii) the Hecataeus Rise has a thick sedimentary rock cover on an indeterminate crust (likely continental) and the crust is significantly thinner than Eratosthenes Seamount based on gravity modelling; (iii) high velocity basement blocks, coincident with highs in the magnetic field, occur in the deep water between Eratosthenes and Hecataeus, and are separated and bounded by deep low-velocity troughs and (iv) one of the high velocity blocks runs parallel to the Cyprus Arc, while the other two appear linked based on the magnetic data and run NW–SE, parallel to the margin of the Hecataeus Rise. The high velocity block beneath the edge of Eratosthenes Seamount is interpreted as an older magmatic intrusion while the linked high velocity blocks along Hecataeus Rise are interpreted as deformed remnant Tethyan oceanic crust or mafic intrusives from the NNW–SSE oriented transform margin marking the northern boundary of Eratosthenes Seamount. Eratosthenes Seamount, the northwestern limit of rifted continental crust from the Levant Margin, is part of a jagged rifted margin transected by transform faults on the northern edge of the lower African Plate that is being obliquely subducted under the Aegean–Anatolian upper plate. The thicker crust of Eratosthenes Seamount may be acting as an asperity on the subducting slab, locally locking up subduction of the Cyprus Arc on its northern margin, while deformed Tethyan oceanic crust remains trapped between its northeastern margin and the Hecataeus Rise.

Description: We present an up-to-date high resolution picture of the ongoing crustal deformation field of Italy, based on an extensive combination of permanent and non-permanent GPS observations carried out since 1994. In addition, we present an updated map of contemporary S Hmax orientations computed by a multidisciplinary data set of well-constrained stress indicators, including both published results and novel analyses. The comparison of stress and geodetic strain-rates directions reveals that both patterns are near-parallel over a large part of the investigated area, highlighting that crustal stress and surface deformation are driven by the same mechanism. The comparison of the azimuthal patterns of surface strain and mantle deformation shows a modest correlation on the Alps and a low correlation along the Apennines chain and the Calabro-Peloritan Arc. Along the Apennines chain, this feature suggests the occurrence of significant strain partitioning and crust–mantle mechanical decoupling. Along the Calabro-Peloritan Arc, the apparent low correlation reflects a different mantle–crust mechanism of deformation to the ongoing subduction and rollback of the Ionian slab. In addition, the superposition of regional/local effects related to second-order sources (crustal lateral density changes, strength contrasts), which at regional/local scale modulate the crustal stress/strain-rate pattern, cannot be ruled out.

Description: A series of linear analysis was performed on the onset of thermal convection of highly compressible fluids, in order to deepen the fundamental insights into the mantle convection of massive super-Earths in the presence of strong adiabatic compression. We consider the temporal evolution (growth or decay) of an infinitesimal perturbation superimposed to a highly compressible fluid which is in a hydrostatic (motionless) and conductive state in a basally heated horizontal layer. As a model of pressure-dependence in material properties, we employed an exponential decrease in thermal expansivity α and exponential increase in (reference) density with depth. The linearized equations for conservation of mass, momentum and internal (thermal) energy are numerically solved for the critical Rayleigh number as well as the vertical profiles of eigenfunctions for infinitesimal perturbations. The above calculations are repeatedly carried out by systematically varying (i) the dissipation number (Di), (ii) the temperature at the top surface and (iii) the magnitude of pressure-dependence in α and . Our analysis demonstrated that the onset of thermal convection is strongly affected by the adiabatic compression, in response to the changes in the static stability of thermal stratification in the fluid layer. For sufficiently large Di where a thick sublayer of stable stratification develops in the layer, for example, the critical Rayleigh number explosively increases with Di, together with drastic decreases in the length scales of perturbations both in vertical and horizontal directions. In particular, for very large Di, a thick ‘stratosphere’ occurs in the fluid layer where the vertical motion is significantly suppressed, resulting in a shrink of the incipient convection in a thin sublayer of unstable thermal stratification. In addition, when Di exceeds a threshold value above which a thermal stratification becomes stable in the entire layer, no perturbation is allowed to grow with time regardless of the Rayleigh number and/or the horizontal wavelength. We also found that the effect of adiabatic compression becomes prominent for higher temperature at the top surface of the fluid layer. These findings may imply the crucial importance of adiabatic compression in understanding the dynamics and evolution of the mantles of massive super-Earths, particularly for those orbiting their parent stars very closely.

Description: In this paper, we propose an approach to compute the coseismic Earth's volume change based on a spherical-Earth elastic dislocation theory. We present a general expression of the Earth's volume change for three typical dislocations: the shear, tensile and explosion sources. We conduct a case study for the 2004 Sumatra earthquake ( M w 9.3), the 2010 Chile earthquake ( M w 8.8), the 2011 Tohoku-Oki earthquake ( M w 9.0) and the 2013 Okhotsk Sea earthquake ( M w 8.3). The results show that mega-thrust earthquakes make the Earth expand and earthquakes along a normal fault make the Earth contract. We compare the volume changes computed for finite fault models and a point source of the 2011 Tohoku-Oki earthquake ( M w 9.0). The big difference of the results indicates that the coseismic changes in the Earth's volume (or the mean radius) are strongly dependent on the earthquakes’ focal mechanism, especially the depth and the dip angle. Then we estimate the cumulative volume changes by historical earthquakes ( M w ≥ 7.0) since 1960, and obtain an Earth mean radius expanding rate about 0.011 mm yr –1 .

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: We present the crustal resistivity structure of the Pamir and Southern Tian Shan orogenic belts at the northwestern promontory of the India–Asia collision zone. The magnetotelluric (MT) data were recorded along a roughly north–south trending, 350 km long corridor from the Pamir Plateau in southern Tajikistan across the Pamir frontal ranges, the Alai Valley and the southwestern Tian Shan to Osh in the Kyrgyz part of the Fergana Basin. In total, we measured at 178 sites, whereof 26 combine broad band and long period recordings. One of the most intriguing features of the 2-D and 3-D inversion results is a laterally extended zone of high electrical conductivity below the Pamir Plateau, with resistivities below 1 m, starting at a depth of ~10–15 km. The high conductivity can be explained with the presence of partially molten rocks at middle to lower crustal levels, possibly related to ongoing migmatization and/or middle/lower crustal flow underneath the Southern Pamir. This interpretation is consistent with a low velocity zone found from local earthquake tomography, relatively high v p / v s ratios, elevated surface heat flow, and thermomechanical modelling suggesting that melting temperatures are reached in the felsic middle crust. In the upper crust of the Pamir and Tian Shan, the Palaeozoic–Mesozoic suture zones appear as electrically conductive, whereas the compact metamorphic rocks of the Muskol-Shatput Dome of the Central Pamir are highly resistive. The intra-montane basin of the Alai Valley—sandwiched between the Pamir and Tian Shan—exhibits a generally conductive upper crust that bifurcates into two conductors at depth. One of them connects to the active Main Pamir Thrust, which is absorbing most of today's convergence between the Pamir and the Tian Shan. Several deeper zones of high conductivity in the middle and lower crust of Central and Northern Pamir likely record fluid release due to metamorphism associated with active continental subduction/delamination.

Description: A new model of the deglaciation history of Antarctica over the past 25 kyr has been developed, which we refer to herein as ICE-6G_C (VM5a). This revision of its predecessor ICE-5G (VM2) has been constrained to fit all available geological and geodetic observations, consisting of: (1) the present day uplift rates at 42 sites estimated from GPS measurements, (2) ice thickness change at 62 locations estimated from exposure-age dating, (3) Holocene relative sea level histories from 12 locations estimated on the basis of radiocarbon dating and (4) age of the onset of marine sedimentation at nine locations along the Antarctic shelf also estimated on the basis of 14 C dating. Our new model fits the totality of these data well. An additional nine GPS-determined site velocities are also estimated for locations known to be influenced by modern ice loss from the Pine Island Bay and Northern Antarctic Peninsula regions. At the 42 locations not influenced by modern ice loss, the quality of the fit of postglacial rebound model ICE-6G_C (VM5A) is characterized by a weighted root mean square residual of 0.9 mm yr –1 . The Southern Antarctic Peninsula is inferred to be rising at 2 mm yr –1 , requiring there to be less Holocene ice loss there than in the prior model ICE-5G (VM2). The East Antarctica coast is rising at approximately 1 mm yr –1 , requiring ice loss from this region to have been small since Last Glacial Maximum. The Ellsworth Mountains, at the base of the Antarctic Peninsula, are inferred to be rising at 5–8 mm yr –1 , indicating large ice loss from this area during deglaciation that is poorly sampled by geological data. Horizontal deformation of the Antarctic Plate is minor with two exceptions. First, O'Higgins, at the tip of the Antarctic Peninsula, is moving southeast at a significant 2 mm yr –1 relative to the Antarctic Plate. Secondly, the margins of the Ronne and Ross Ice Shelves are moving horizontally away from the shelf centres at an approximate rate of 0.8 mm yr –1 , in viscous response to the early Holocene unloading of ice from the current locations of the ice shelf centers. ICE-6G_C (VM5A) fits the horizontal observations well (wrms residual speed of 0.7 mm yr –1 ), there being no need to invoke any influence of lateral variation in mantle viscosity. ICE-6G_C (VM5A) differs in several respects from the recently published W12A model of Whitehouse et al. First, the upper-mantle viscosity in VM5a is 5 10 20 Pa s, half that in W12A. The VM5a profile, which is identical to that inferred on the basis of the Fennoscandian relaxation spectrum, North American relative sea level histories and Earth rotation constraints, when coupled with the revised ICE-6G_C deglaciation history, fits all of the available constraints. Secondly, the net contribution of Antarctica ice loss to global sea level rise is 13.6 m, 2/3 greater than the 8 m in W12A. Thirdly, ice loss occurs quickly from 12 to 5 ka, and the contribution to global sea level rise during Meltwater Pulse 1B (11.5 ka) is large (5 m), consistent with sedimentation constraints from cores from the Antarctica ice shelf. Fourthly, in ICE-6G_C there is no ice gain in the East Antarctica interior, as there is in W12A. Finally, the new model of Antarctic deglaciation reconciles the global constraint upon the global mass loss during deglaciation provided by the Barbados record of relative sea level history when coupled with the Northern Hemisphere counterpart of this new model.

Description: The Himalaya is the result of the on-going convergence and collision of India and Asia. The internal configuration and processes that govern the rise of the Himalayan Mountains and Tibetan Plateau are crucial to understand continental collision zones. However, knowledge of the prior configuration of the colliding plates is equally important, since inherited (pre-orogenic/basement) structures can undeniably influence the development of the orogenic architecture throughout the orogen's cycle of collision and eventual collapse. Three northeast-trending palaeotopographic ridges of faulted Precambrian Indian basement underlie the Ganga basin south of the Himalaya. Our paper illustrates a crustal-scale fault origin for these ridges and succeeds in determining how far north beneath the Himalayan system they extend and how they ultimately govern the location of upper crustal faults in southern Tibet. Spectrally filtered EGM2008 Bouguer gravity data and edges in its horizontal gradient at different source depths (‘gravity worms’) over northern Peninsular India, the Himalaya and southern Tibet reveal several continuous Himalayan cross-strike discontinuities interpreted to represent crustal faults. Gravity lineaments in Peninsular India coincide with edges of the Precambrian basement ridges and megakinks up to 100 km wide develop in foreland cover sequences between the interpreted basement faults. The interpreted basement faults project northward beneath the Himalayan system and southern Tibet. Our results suggest that several active Himalayan cross-strike faults, such as the ones related to many graben in southern Tibet, are rooted in the underplated Indian lower crust or step en échelon along interpreted basement faults. Our interpretation thus suggests that south Tibet graben are spatially related to deep-seated crustal-scale faults rooted in the underplated Indian crust. These major discontinuities partition the Himalayan range into distinct zones, and could ultimately contribute to lateral variability in tectonic evolution along the orogen's strike.

Description: We explore the impact of deep ductile shear zones on post-seismic deformation following a finite length strike-slip earthquake. We show that the pattern of post-seismic vertical surface deformation surrounding the fault is a discriminant for the existence of high viscosities immediately below the seismogenic layer, regardless of whether the model contains purely distributed creep or also includes a component of localized creep at subseismogenic depths. Post-seismic deformation characterized by initially fast relaxation followed by a slower relaxation is predicted by models that include both localized creep in a subseismogenic shear zone and distributed creep in the surrounding region, even if they only contain steady Maxwell viscoelasticity. This post-seismic deformation is similar to that in models that approximate the ductile lithosphere and/or asthenosphere with Burgers viscoelasticity. We find that the post-seismic deformation following the 1997 M w 7.6 Manyi, China, earthquake, is consistent with a post-seismic model composed of a lower Maxwell viscoelastic region with viscosity 10 19 Pa s and a 5 km wide, Maxwell viscoelastic shear zone with viscosity 10 18 Pa s beneath the fault.

Description: The Amundsen Sea Embayment of West Antarctica represents a key component in the tectonic history of Antarctic–New Zealand continental breakup. The region played a major role in the plate-kinematic development of the southern Pacific from the inferred collision of the Hikurangi Plateau with the Gondwana subduction margin at approximately 110–100 Ma to the evolution of the West Antarctic Rift System. However, little is known about the crustal architecture and the tectonic processes creating the embayment. During two ‘RV Polarstern’ expeditions in 2006 and 2010 a large geophysical data set was collected consisting of seismic-refraction and reflection data, ship-borne gravity and helicopter-borne magnetic measurements. Two P -wave velocity–depth models based on forward traveltime modelling of nine ocean bottom hydrophone recordings provide an insight into the lithospheric structure beneath the Amundsen Sea Embayment. Seismic-reflection data image the sedimentary architecture and the top-of-basement. The seismic data provide constraints for 2-D gravity modelling, which supports and complements P -wave modelling. Our final model shows 10–14-km-thick stretched continental crust at the continental rise that thickens to as much as 28 km beneath the inner shelf. The homogenous crustal architecture of the continental rise, including horst and graben structures are interpreted as indicating that wide-mode rifting affected the entire region. We observe a high-velocity layer of variable thickness beneath the margin and related it, contrary to other ‘normal volcanic type margins’, to a proposed magma flow along the base of the crust from beneath eastern Marie Byrd Land—West Antarctica to the Marie Byrd Seamount province. Furthermore, we discuss the possibility of upper mantle serpentinization by seawater penetration at the Marie Byrd Seamount province. Hints of seaward-dipping reflectors indicate some degree of volcanism in the area after break-up. A set of gravity anomaly data indicate several phases of fully developed and failed rift systems, including a possible branch of the West Antarctic Rift System in the Amundsen Sea Embayment.

Description: Geophysical data are the main source of information about the subsurface. Geophysical techniques are, however, highly non-unique in determining specific physical parameters and boundaries of subsurface objects. To obtain actual physical information, an inversion process is often applied, in which measurements at or above the Earth surface are inverted into a 2- or 3-D subsurface spatial distribution of the physical property. Interpreting these models into structural objects, related to physical processes, requires a priori knowledge and expert analysis which is susceptible to subjective choices and is therefore often non-repeatable. In this research, we implemented a recently introduced object-based approach to interpret the 3-D inversion results of a single geophysical technique using the available a priori information and the physical and geometrical characteristics of the interpreted objects. The introduced methodology is semi-automatic and repeatable, and allows the extraction of subsurface structures using 3-D object-oriented image analysis (3-D OOA) in an objective knowledge–based classification scheme. The approach allows for a semi-objective setting of thresholds that can be tested and, if necessary, changed in a very fast and efficient way. These changes require only changing the thresholds used in a so-called ruleset, which is composed of algorithms that extract objects from a 3-D data cube. The approach is tested on a synthetic model, which is based on a priori knowledge on objects present in the study area (Tanzania). Object characteristics and thresholds were well defined in a 3-D histogram of velocity versus depth, and objects were fully retrieved. The real model results showed how 3-D OOA can deal with realistic 3-D subsurface conditions in which the boundaries become fuzzy, the object extensions become unclear and the model characteristics vary with depth due to the different physical conditions. As expected, the 3-D histogram of the real data was substantially more complex. Still, the 3-D OOA-derived objects were extracted based on their velocity and their depth location. Spatially defined boundaries, based on physical variations, can improve the modelling with spatially dependent parameter information. With 3-D OOA, the non-uniqueness on the location of objects and their physical properties can be potentially significantly reduced.

Description: We estimate Eurasia-North America Plate motion rotations at ~1-Myr intervals for the past 20 Myr from more than 11 000 crossings of 21 magnetic reversals from Chron 1n (0.78 Ma) to C6no (19.72 Ma) and flow lines digitized from the Charlie Gibbs, Bight and Molloy fracture zones and transform faults. Adjusted for outward displacement, the 21 best-fitting rotations determined from a simultaneous inversion of the numerous kinematic data reconstruct the reversal crossings with weighted root mean square misfits of only 1–2 km and 0.2–7 km for the transform fault and fracture zone crossings. The new rotations clearly define a ~1000 km southward shift of the rotation pole and 20 per cent slowdown in seafloor spreading rates between 7 and 6 Ma, preceded by apparently steady plate motion from 19.7 to ~7 Ma. Data for times since C3An.2 (6.7 Ma) are well fit by a stationary pole of rotation and constant rate of angular opening, consistent with steady motion since 6.7 Ma. The southward shift of the rotation pole at 7–6 Ma implies that Eurasia-North America motion in northeastern Asia changed from slowly convergent before 7 Ma to slowly divergent afterward. Crossings of magnetic reversals C1n through C3An.1 (6.0 Ma) are well fit everywhere in the Arctic basin and south to the Azores triple junction, indicating that the Eurasia and North America plates have not deformed along their mutual boundary since at least 6.0 Ma. However, the new rotations systematically overrotate magnetic lineations older than C3An.1 (6.0 Ma) within 200 km of the Azores triple junction and also overrotate lineations older than C5n along the Gakkel Ridge in the Arctic Basin. Barring misidentifications of the magnetic anomalies in those areas, the pattern and magnitude of the systematic misfits imply that slow (~1 mm yr –1 ) distributed or microplate deformation occurred in one or both regions.

Description: Here we inverted the GPS data to infer the coseismic slip of the Tohoku-Oki earthquake and the time-dependent afterslip distribution in the 4 months following the main shock. The Tohoku-Oki earthquake showed an unexpected magnitude and a characteristic depth-dependent differentiation of seismic energy radiation. In this context the estimation and comparison of the distribution of the fault portions that slip coseismically and post-seismically contribute to a better understanding of the variation of frictional characteristics of the plate interface. The inferred coseismic slip extends in a relatively compact region located updip from the hypocentre and reaches its highest value (about 60 m) near the trench. Afterslip occurs mostly outside the coseismic rupture and is distributed in two main modal centres. It reaches its largest values in an area located downdip of the coseismic slip and extends to a depth of 80 km. In the depth range between 30 and 50 km afterslip overlaps the portion of the fault that experienced historical moderate earthquakes, high-frequency seismic radiation and thrust-type aftershocks. The behaviour of this area can be explained by a rheologically heterogeneous region made of a ductile fault matrix interspersed with compact brittle asperities. On the contrary, the region beneath 50–60 km depth is probably characterized by a fully velocity strengthening behaviour. Southern afterslip, located off-Chiba Prefecture, is probably related to the M w 7.9 Ibaraki-Oki aftershock. The northward extension of the afterslip stops at a latitude of about 40°N, just south of the off-Aomori region. This may be related to three large events occurred in this area during the last century and the consequent strong coupling or complete depletion of the accumulated strain that characterize this region.

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 present a revised interpretation of magnetic anomalies and fracture zones on the Southwest Indian Ridge (SWIR; Africa–Antarctica) and the Southeast Indian Ridge (SEIR; Capricorn–Antarctica) and use them to calculate 2-plate finite rotations for anomalies 34 to 20 (84 to 43 Ma). Central Indian Ridge (CIR; Capricorn–Africa) rotations are calculated by summing the SWIR and SEIR rotations. These rotations provide a high-resolution record of changes in the motion of India and Africa at the time of the onset of the Reunion plume head. An analysis of the relative velocities of India, Africa and Antarctica leads to a refinement of previous observations that the speedup of India relative to the mantle was accompanied by a slowdown of Africa. The most rapid slowdown of Africa occurs around Chron 32Ay (71 Ma), the time when India's motion relative to Africa notably starts to accelerate. Using the most recent Geomagnetic Polarity Timescale (GTS12) we show that India's velocity relative to Africa was characterized by an acceleration from roughly 60 to 180 mm yr –1 between 71 and 66 Ma, a short pulse of superfast motion (~180 mm yr –1 ) between 66 and 63 Ma, an abrupt slowdown to 120 mm yr –1 between 63 and 62 Ma, and then a long period (63 to 47 Ma) of gradual slowing, but still fast motion (~100 mm yr –1 ), which ends with a rapid slowdown after Chron 21o (47 Ma). Changes in the velocities of Africa and India with respect to the mantle follow a similar pattern. The fastest motion of India relative to the mantle, ~220 mm yr –1 , occurs during Chron 29R. The SWIR rotations constrain three significant changes in the migration path of the Africa–Antarctic stage poles: following Chron 33y (73 Ma), following Chron 31y (68 Ma), and following Chron 24o (54 Ma). The change in the migration path of the SWIR stage poles following Chron 33y is coincident with the most rapid slowdown in Africa's motion. The change in the migration path after Chron 31y, although coincident with the most rapid acceleration of India's northward motion, may be related to changes in ridge push forces on the SWIR associated with the onset of extension along the Bain transform fault zone. The initial slowdown in India's motion relative to Africa between 63 and 62 Ma is more abrupt than predictions based on published plume head force models, suggesting it might have been caused by a change in plate boundary forces. The abrupt change in the migration path of the SWIR stage poles after Chron 24o is not associated with major changes in the velocities of either Africa or India and may reflect Atlantic basin plate motion changes associated with the arrival at the Earth's surface of the Iceland plume head. The abruptness of India's slowdown after Chron 21o is consistent with a collision event.

Description: Thin plate flexure theory provides an accurate model for the response of the lithosphere to vertical loads on horizontal length scales ranging from tens to hundreds of kilometres. Examples include flexure at seamounts, fracture zones, sedimentary basins and subduction zones. When applying this theory to real world situations, most studies assume a locally uniform plate thickness to enable simple Fourier transform solutions. However, in cases where the amplitude of the flexure is prominent, such as subduction zones, or there are rapid variations in seafloor age, such as fracture zones, these models are inadequate. Here we present a computationally efficient algorithm for solving the thin plate flexure equation for non-uniform plate thickness and arbitrary vertical load. The iterative scheme takes advantage of the 2-D fast Fourier transform to perform calculations in both the spatial and spectral domains, resulting in an accurate and computationally efficient solution. We illustrate the accuracy of the method through comparisons with known analytic solutions. Finally, we present results from three simple models demonstrating the differences in trench outer rise flexure when 2-D variations in plate rigidity and applied bending moment are taken into account. Although we focus our analysis on ocean trench flexure, the method is applicable to other 2-D flexure problems having spatial rigidity variations such as seamount loading of a thermally eroded lithosphere or flexure across the continental–oceanic crust boundary.

Description: Theory has been long established for computing the elastic response of a spherically symmetric terrestrial planetary body to both body tide and surface loading forces. However, for a planet with laterally heterogeneous mantle structure, the response is usually computed using a fully numerical approach. In this paper, we develop a semi-analytic method based on perturbation theory to solve for the elastic response of a planetary body with lateral heterogeneities in its mantle. We present a derivation of the governing equations for our second-order perturbation method and use them to study the high-order tidal effects caused by mode coupling between degree-2 body tide forcing and the laterally heterogeneous elastic structure of the mantle. We test our method by applying it to the Moon in which small long-wavelength lateral heterogeneities are assumed to exist in the elastic moduli of the lunar mantle. The tidal response of the Moon is determined mode by mode, for lateral heterogeneities with different depth ranges within the mantle and different horizontal scales. Our perturbation method solutions are compared with numerical results, showing remarkable agreement between the two methods. We conclude that our perturbation method provides accurate results and can be adapted to address a variety of forward and inverse response problems.

Description: Teleseismic data recorded at 13 broad-band seismological stations across northwest part of the Tethyan Himalaya and eastern Ladakh are analysed to determine the seismic characteristics of the crust and upper mantle beneath the northwest India–Asia collision zone. The receiver functions computed from teleseismic P- waveform for a wide range of backazimuth show strong azimuthal variation in the Indus suture zone (ISZ), the zone which marks the collision and subsequent subduction of both the Tethyan oceanic plate and Indian continental plate beneath Eurasia. The teleseismic waves piercing the ISZ do not show clear P -to- S ( Ps ) converted phase at the depth of Moho. In contrast, the waves piercing the Karakoram zone, Ladakh batholith and the Tethyan Himalayan region south of the ISZ clearly show the Moho converted Ps phase and corresponding inverted models reveal variation of crustal thickness from ~60 km beneath the Tethyan Himalaya to ~80 km beneath the Karakoram fault zone. A prominent intracrustal low velocity zone (IC-LVZ) is detected in the shear wave velocity models within the depth range ~15–40 km. The IC-LVZ identified at the stations both north and south of the ISZ can be interpreted as due to presence of fluid/partial melt. Our study provides compelling evidence that the mid-crustal low velocity zone does extend across the suture zone, in to the Tethyan Himalaya. The contact between this serpentinized ultramafic rocks and the eclogitized Indian continental crust in the suture zone is identified at ~47–50 km depth.

Description: The Tien Shan is the largest active intracontinental orogenic belt on Earth. To better understand the processes causing mountains to form at great distances from a plate boundary, we analyse passive source seismic data collected on 40 broad-band stations of the MANAS project (2005–2007) and 12 stations of the permanent KRNET seismic network to determine variations in crustal thickness and shear wave speed across the range. We jointly invert P - and S -wave receiver functions with surface wave observations from both earthquakes and ambient noise to reduce the ambiguity inherent in the images obtained from the techniques applied individually. Inclusion of ambient noise data improves constraints on the upper crust by allowing dispersion measurements to be made at shorter periods. Joint inversion can also reduce the ambiguity in interpretation by revealing the extent to which various features in the receiver functions are amplified or eliminated by interference from multiples. The resulting wave speed model shows a variation in crustal thickness across the range. We find that crustal velocities extend to ~75 km beneath the Kokshaal Range, which we attribute to underthrusting of the Tarim Basin beneath the southern Tien Shan. This result supports the plate model of intracontinental convergence. Crustal thickness elsewhere beneath the range is about 50 km, including beneath the Naryn Valley in the central Tien Shan where previous studies reported a shallow Moho. This difference apparently is the result of wave speed variations in the upper crust that were not previously taken into account. Finally, a high velocity lid appears in the upper mantle of the Central and Northern part of the Tien Shan, which we interpret as a remnant of material that may have delaminated elsewhere under the range.

Description: Errors in the satellite orbits are considered to be a limitation for Interferometric Synthetic Aperture Radar (InSAR) time-series techniques to accurately measure long-wavelength (〉50 km) ground displacements. Here we examine how orbital errors propagate into relative InSAR line-of-sight velocity fields and evaluate the contribution of orbital errors to the InSAR uncertainty. We express the InSAR uncertainty due to the orbital errors in terms of the standard deviations of the velocity gradients in range and azimuth directions (range and azimuth uncertainties). The range uncertainty depends on the magnitude of the orbital errors, the number and time span of acquisitions. Using reported orbital uncertainties we find range uncertainties of less than 1.5 mm yr –1  100 km –1 for ERS, less than 0.5 mm yr –1  100 km –1 for Envisat and ~0.2 mm yr –1  100 km –1 for TerraSAR-X and Sentinel-1. Under a conservative scenario, we find azimuth uncertainties of better than 1.5 mm yr –1  100 km –1 for older satellites (ERS and Envisat) and better than 0.5 mm yr –1  100 km –1 for modern satellites (TerraSAR-X and Sentinel-1). We validate the expected uncertainties using LOS velocity fields obtained from Envisat SAR imagery. We find residual gradients of 0.8 mm yr –1  100 km –1 or less in range and of 0.95 mm yr –1  100 km –1 or less in azimuth direction, which fall within the 1 to 2 uncertainties. The InSAR uncertainties due to the orbital errors are significantly smaller than generally expected. This shows the potential of InSAR systems to constrain long-wavelength geodynamic processes, such as continent-scale deformation across entire plate boundary zones.

Description: A method for subsurface recognition of blind geological bodies is presented using combined surface constraints and 3-D structural modelling that incorporates constraints from detailed mapping, and potential-field inversion modelling. This method is applied to the Mount Painter Province and demonstrates that addition of low density material is required to reconcile the gravity signature of the region. This method may be an effective way to construct 3-D models in regions of excellent structural control, and can be used to assess the validity of surface structures with 3-D architecture. Combined geological and potential-field constrained inversion modelling of the Mount Painter Province was conducted to assess the validity of the geological models of the region. Magnetic susceptibility constrained stochastic property inversions indicates that the northeast to southwest structural trend of the relatively magnetic meta-sedimentary rocks of the Radium Creek Group in the Mount Painter Inlier is reconcilable with the similar, northeast to southwest trending positive magnetic anomalies in the region. Radium Creek Group packages are the major contributor of the total magnetic response of the region. However field mapping and the results of initial density constrained stochastic property inversion modelling do not correlate with a large residual negative gravity anomaly central to the region. Further density constrained inversion modelling indicates that an additional large body of relatively low density material is needed within the model space to account for this negative density anomaly. Through sensitivity analysis of multiple geometrical and varied potential-field property inversions, the best-fitting model records a reduction in gravity rms misfit from 21.9 to 1.69 mGal, representing a reduction from 56 to 4.5 per cent in respect to the total dynamic range of 37.5 mGal of the residual anomaly. This best-fitting model incorporates a volumetrically significant source body of interpreted felsic, low density material (10 12 m 3 ) impinging on the central-west of the Mount Painter Inlier and overlying Neoproterozoic sequences, and the emplacement of more mafic affinities in the northeast and east. The spatial association and circular geometry of these granitoid bodies suggests an affinity with the Palaeozoic ~460–440 Ma British Empire Granite that outcrops in the Mount Painter Inlier. The intrusion of this additional material in the Palaeozoic could either be the product of; or contributed to, an increased local geotherm and heat flow in the region during the Palaeozoic.

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: The Pacific and Australian plates in the South Island, New Zealand (NZ) converge at a rate of about 4 cm yr –1 . Accommodation of the continental part of this convergence in the lithospheric mantle is both poorly understood and currently controversial yet it is a problem of fundamental importance for understanding lithospheric thickening. End-member possibilities range from the classical model of asymmetric subduction to symmetric viscous thickening. Seismic tomography has the potential to image this process. However, tomographic images to date are poorly constrained due to the lack of appropriate earthquakes. Improved teleseismic tomography of the region has been achieved by increasing data coverage and applying a novel scheme of correcting for crustal structure by ray tracing through a newly created model of shallow shear wave velocity derived from the inversion of noise-based dispersion measurements. Our resulting models suggest the lithospheric mantle high velocities at the continental plate boundary extend no deeper than approximately 125 km, evidence against both previous models of viscous drip and typical asymmetric subduction zones. This high velocity core extends from north to south along the axis of South Island suggesting that mantle convergence is accommodated along the older, mid-Cenozoic, plate boundary. West of South Island, a high velocity west dipping zone may define the remnant Cretaceous subduction zone that has been distorted by Cenozoic transcurrent deformation. We present our new 3-D seismic velocity models together with a compatible tectonic model and discuss their implications for the nature of lithospheric evolution at this convergent boundary.

Description: We use continuous GPS measurements from 31 stations in southern Mexico to model coseismic slip and post-seismic deformation from the 2012 March 20 M w  = 7.5 Ometepec earthquake, the first large thrust earthquake to occur below central Mexico during the modern GPS era. Coseismic offsets ranging from ~280 mm near the epicentre to 5 mm or less at sites far from the epicentre are fit best by a rupture focused between ~15 and 35 km depth, consistent with an independent seismological estimate. The corresponding geodetic moment of 1.4 10 20 N·m is within 10 per cent of two independent seismic estimates. Transient post-seismic motion recorded by GPS sites as far as 300 km from the rupture has a different horizontal deformation gradient and opposite sense of vertical motion than do the coseismic offsets. A forward model of viscoelastic relaxation as a result of our new coseismic slip solution incorrectly predicts uplift in areas where post-seismic subsidence was recorded and indicates that viscoelastic deformation was no more than a few per cent of the measured post-seismic deformation. The deformation within 6 months of the earthquake was thus strongly dominated by fault afterslip. The post-seismic GPS time-series are well fit as logarithmically decaying fault afterslip on an area of the subduction interface up to 10 times larger than the earthquake rupture zone, extending as far as 220 km inland. Afterslip had a cumulative geodetic moment of 2.0 10 20 N·m, ~40 per cent larger than the Ometepec earthquake. Tests for the shallow and deep limits for the afterslip require that it included much of the earthquake rupture zone as well as regions of the subduction interface where slow slip events and non-volcanic tremor have been recorded and areas even farther downdip on the flat interface. Widespread afterslip below much of central Mexico suggests that most of the nearly flat subduction interface in this region is conditionally stable and thus contributes measurable transient deformation to large areas of Mexico south of and in the volcanic belt.

Description: We have derived a shallow subsurface 2-D tomographic P -wave velocity image of the Deccan Volcanic Province (DVP) of India using first-arrival traveltime data along a 90-km-long N–S trending seismic profile in the Deccan Syneclise region. The tomographic image depicts smooth velocity variations of Quaternary and Tertiary (2.0–3.0 km s –1 ) sediments, basalts/traps (5.0–5.5 km s –1 ), sub-trappean Mesozoic sediments (4.3–4.5 km s –1 ) as well as the basement (5.9–6.1 km s –1 ) geometry down to a maximum depth of 5.0 km. Due to Late Cretaceous volcanism and outpouring of basaltic lava flows, this region is affected by numerous dyke intrusions and thick basaltic trap (2–3 km) exposed on the surface and surrounded by graben structures due to deep basinal faults forming a large igneous province. Although sub-basalt imaging is a major challenge for the oil industry, with the help of tomographic imaging technique of first-arrival seismic refraction data, we were able to image sub-trappean Mesozoic sediments (〈0.75 km) deposited below the two sequences of thick basaltic flows above the basement. The imaged Mesozoic sediments are expected to contain hydrocarbon because of their wide extension in this sedimentary basin with suitable trapping mechanism due to basalts. The robustness of the velocity image is assessed through numerous tests like velocity perturbations, 2 estimates, rms residuals of traveltime fit, uncertainty estimates through computation of ray-density or hits and series of checkerboard resolution tests with velocity anomalies having different cell size. The thickness of the basalt and the sub-trappean Mesozoic sediments along with the basement geometry obtained from tomography are constrained through ray-trace modelling and pre-stack depth migration (PSDM) of the wide-angle reflection phases for different shot gathers along the profile.

Description: Numerical experiments of convection with grain-damage are used to develop scaling laws for convective heat flow, mantle velocity and plate velocity across the stagnant lid and plate-tectonic regimes. Three main cases are presented in order of increasing complexity: a simple case wherein viscosity is only dependent on grain size, a case where viscosity depends on temperature and grain size, and finally a case where viscosity is temperature and grain size sensitive, and the grain-growth (or healing) is also temperature sensitive. In all cases, convection with grain-damage scales differently than Newtonian convection; whereas the Nusselt number (Nu), typically scales with the reference Rayleigh number, Ra 0 , to the 1/3 power, for grain-damage this exponent is larger because increasing Ra 0 also enhances damage. In addition, Nu, mantle velocity, and plate velocity are also functions of the damage to healing ratio, ( D / H ); increasing D / H increases Nu because more damage leads to more vigorous convection. For the fully realistic case, numerical results show stagnant lid convection, fully mobilized convection that resembles the temperature-independent viscosity case, and partially mobile or transitional convection, depending on D / H , Ra 0 , and the activation energies for viscosity and healing. Applying our scaling laws for the fully realistic case to Earth and Venus we demonstrate that increasing surface temperature dramatically decreases plate speed and heat flow, essentially shutting down plate tectonics, due to increased healing in lithospheric shear zones, as proposed previously. Contrary to many previous studies, the transitional regime between the stagnant lid and fully mobilized regimes is large, and the transition from stagnant lid to mobile convection is gradual and continuous. Thus planets could exhibit a full range of surface mobility, as opposed to the bimodal distribution of fully mobile lid planets and stagnant lid planets that is typically assumed.

Description: Three magnetotelluric (MT) profiles in northwestern Canada cross the central and western segments of Great Slave Lake shear zone (GSLsz), a continental scale strike-slip structure active during the Slave-Rae collision in the Proterozoic. Dimensionality analysis indicates that (i) the resistivity structure is approximately 2-D with a geoelectric strike direction close to the dominant geological strike of N45°E and that (ii) electrical anisotropy may be present in the crust beneath the two southernmost profiles. Isotropic and anisotropic 2-D inversion and isotropic 3-D inversions show different resistivity structures on different segments of the shear zone. The GSLsz is imaged as a high resistivity zone (〉5000  m) that is at least 20 km wide and extends to a depth of at least 50 km on the northern profile. On the southern two profiles, the resistive zone is confined to the upper crust and pierces an east-dipping crustal conductor. Inversions show that this dipping conductor may be anisotropic, likely caused by conductive materials filling a network of fractures with a preferred spatial orientation. These conductive regions would have been disrupted by strike-slip, ductile deformation on the GSLsz that formed granulite to greenschist facies mylonite belts. The pre-dominantly granulite facies mylonites are resistive and explain why the GSLsz appears as a resistive structure piercing the east-dipping anisotropic layer. The absence of a dipping anisotropic/conductive layer on the northern MT profile, located on the central segment of the GSLsz, is consistent with the lack of subduction at this location as predicted by geological and tectonic models.

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 3-D shear velocity structure beneath South India's Dharwar Craton determined from fundamental mode Rayleigh waves phase velocities reveals the existence of anomalously high velocity materials in the depth range of 50–100 km. Tomographic analysis of seismograms recorded on a network of 35 broad-band seismographs shows the uppermost mantle shear wave speeds to be as high as 4.9 km s –1 in the northwestern Dharwar Craton, decreasing both towards the south and the east. Below ~100 km, the shear wave speed beneath the Dharwar Craton is close to the global average shear wave speed at these depths. Limitations of usable Rayleigh phase periods, however, have restricted the analysis to depths of 120 km, precluding the delineation of the lithosphere–asthenosphere boundary in this region. However, pressure–temperature analysis of xenoliths in the region suggests a lithospheric thickness of at least ~185 km during the mid-Proterozoic period. The investigations were motivated by a search for seismic indicators in the shallow mantle beneath the distinctly different parts of the Dharwar Craton otherwise distinguished by their lithologies, ages and crustal structure. Since the ages of cratonic crust and of the associated mantle lithosphere around the globe have been found to be broadly similar and their compositions bimodal in time, any distinguishing features of the various parts of the Dharwar shallow mantle could thus shed light on the craton formation process responsible for stabilizing the craton during the Meso- and Neo-Archean.

Description: Frontier hydrocarbon development projects in the deepwater slopes of the Gulf of Mexico Basin, Santos Basin and Lower Congo Basin all require wells to cross ductile layers of autochthonous or allochthonous salt moving at peak rates of 100 mm yr –1 . The Couette–Poiseuille number is introduced here to help pinpoint the depth of shear stress reversal in such salt layers. For any well-planned through salt, the probable range of creep forces of moving salt needs to be taken into account when designing safety margins and load-factor tolerance of the well casing. Drag forces increase with wellbore diameter, but more significantly with effective viscosity and speed of the creeping salt layer. The potential drag forces on cased wellbores in moving salt sheets are estimated analytically using a range of salt viscosities (10 15 –10 19 Pa s) and creep rates (0–10 mm yr –1 ). Drag on perfectly rigid casing of infinite strength may reach up to 13 Giga Newton per meter wellbore length in salt having a viscosity of 10 19 Pa s. Well designers may delay stress accumulations due to salt drag when flexible casing accommodates some of the early displacement and strain. However, all creeping salt could displace, fracture and disconnect well casing, eventually. The shear strength of typical heavy duty well casing (about 1000 MPa) can be reached due to drag by moving salt. Internal flow of salt will then fracture the casing near salt entry and exit points, but the structural damage is likely to remain unnoticed early in the well-life when the horizontal shift of the wellbore is still negligibly small (at less than 1 cm yr –1 ). Disruption of casing and production flow lines within the anticipated service lifetime of a well remains a significant risk factor within distinct zones of low-viscosity salt which may reach ultrafast creep rates of 100 mm yr –1 .

Description: The Antarctic Peninsula (AP) consists of a long lived and uniquely well preserved magmatic arc system. The broad tectonic structure of the AP arc is well understood. However, magmatic processes occurring along the arc are only constrained by regional geophysical and relatively sparse geological data. Key questions remain about the timing, volume, and structural controls on magma emplacement. We present new high resolution aeromagnetic data across Adelaide Island, on the western margin of the AP revealing the complex structure of the AP arc/forearc boundary. Using digital enhancement, 2-D modelling and 3-D inversion we constrain the form of the magnetic sources at the arc/forearc boundary. Our interpretation of these magnetic data, guided by geological evidence and new zircon U-Pb dating, suggests significant Palaeogene to Neogene magmatism formed ~25 per cent of the upper crust in this region (~7500 km 3 ). Significant structural control on Neogene magma emplacement along the arc/forearc boundary is also revealed. We hypothesize that this Neogene magmatism reflects mantle return flow through a slab window generated by Late Palaeogene cessation of subduction south of Adelaide Island. This mantle process may have affected the final stages of arc magmatism along the AP margin.

Description: The intrusion mechanism and internal structure of sills are still under debate. We present a detailed magnetic study, including anisotropy of magnetic susceptibility and rock magnetic analyses of a Cretaceous (94 Ma), 7-m-thick sill from the Lusitanian Basin in Portugal, the Foz da Fonte sill. The results, from both the top surface and a vertical profile, allow us to propose a model for the magmatic flow pattern and sense of flow. According to their location in the vertical profile, three magnetic fabric domains are identified: (1) at the borders, qualified as chilled margins (~0–50 cm), low anisotropies suggest that low velocity gradients and heterogeneous flow paths occurred during the initial emplacement stages; (2) in the centre of the sill, where low anisotropies are observed, low shear gradients and magma displacement close to pure translation is inferred and (3) in the intermediate zones, high anisotropy values are ascribed to zones having undergone high shear gradients. The mean magnetic lineations from the top surface and basal contact indicate an almost horizontal and NW–SE orientation (azimuth: 310°) which agrees with the preferred orientation of iron oxide grain clusters and with the elongation of vesicles considered as coaxial with the magma flow direction. Moreover, the magnetic foliation planes and the lineations show both a mirror imbrication relative to the average upper and lower border surfaces of the sill, pointing to a flow direction towards the SE. Based on these results and on the interpretation of two seismic reflection lines, we show that the Cabo Raso magnetic anomaly, located 25 km to NW of the FF-sill, is associated to Cretaceous magmatic intrusions from which the sill likely originated. This tectono-magmatic setting is discussed with respect to the West Iberia Late Cretaceous magmatism, integrating magnetic anomalies, isotope chronology and tectonics.

Description: Southern Mendoza and northern Neuquén Provinces, south of the Pampean Shallow Subduction region in western Argentina, are host to the 〈2 Myr Payunia Basaltic Province, which covers ~39 500 km 2 with primarily basaltic intraplate volcanism. This backarc igneous province can be explained by extension due to trench roll-back following steepening of a flat slab that existed in the middle to late Miocene. Magnetotelluric data collected at 37 sites from 67°W to 70°W and 35°S to 38°S are used to probe the source of the Payún Matrú basalts. These data, which require significantly 3-D structure, are inverted with a 3-D non-linear conjugate gradient algorithm that minimizes structure for a given data misfit. We identify two significant electrically conductive structures. One, called the SWAP (shallow western asthenospheric plume) approaches the surface beneath the Payún Matrú Caldera and the Trómen Volcano and dips westward towards the subducted Nazca slab. The second, called the DEEP (deep eastern plume) approaches the surface ~100 km to the southeast of Payún Matrú and dips steeply east to ~400 km depth while remaining above the subducted Nazca slab. We use a variety of model assessment techniques including forward modelling and constrained inversion to test the veracity of these features. We interpret the SWAP as the source of the 〈2 Myr intraplate volcanism. Our model assessment permits but does not require the SWAP to connect to the Nazca slab. The SWAP and DEEP are electrically connected only in the shallow crust, which is likely due to the Neuquén sedimentary basin and not a magmatic process. We propose that the SWAP and DEEP may have been more robustly connected in the past, but that the DEEP was decapitated to form the SWAP when shallow northwestward mantle flow resumed during steepening of the slab. The ~2 Myr basaltic volcanism is the result of this decapitated DEEP magma that had ponded below the crust until extension allowed eruption. The westward dip of the SWAP is interpreted to be the result of shear in the renewed mantle corner flow—this explains why the SWAP and Nazca slab can appear connected, yet there is no recent arc-signature magma in this region.

Description: This study presents the results of a deep seismic survey across the north Algerian margin, based on the combination of 2-D multichannel and wide-angle seismic data simultaneously recorded by 41 ocean bottom seismometers deployed along a north–south line extending 180 km off Jijel into the Algerian offshore basin, and 25 land stations deployed along a 100-km-long line, cutting through the Lesser Kabylia and the Tellian thrust-belt. The final model obtained using forward modelling of the wide-angle data and pre-stack depth migration of the seismic reflection data provides an unprecedented view of the sedimentary and crustal structure of the margin. The sedimentary layers in the Algerian basin are 3.75 km thick to the north and up to 4.5–5 km thick at the foot of the margin. They are characterized by seismic velocities from 1.9 to 3.8 km s –1 . Messinian salt formations are about 1 km thick in the study area, and are modelled and imaged using a velocity between 3.7 and 3.8 km s –1 . The crust in the deep sea basin is about 4.5 km thick and of oceanic origin, presenting two distinct layers with a high gradient upper crust (4.7–6.1 km s –1 ) and a low gradient lower crust (6.2–7.1 km s –1 ). The upper-mantle velocity is constrained to 7.9 km s –1 . The ocean–continent transition zone is very narrow between 15 and 20 km wide. The continental crust reaches 25 km thickness as imaged from the most landward station and thins to 5 km over a less than 70 km distance. The continental crust presents steep and asymmetric upper- and lower-crustal geometry, possibly due to either asymmetric rifting of the margin, an underplated body, or flow of lower crustal material towards the ocean basin. Present-time deformation, as imaged from three additional seismic profiles, is characterized by an interplay of gravity-driven mobile-salt creep and active thrusting at the foot of the tectonically inverted Algerian margin.

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.

Description: For the first time, a deep seismic data set acquired in the frame of the Algerian–French SPIRAL program provides new insights regarding the origin of the westernmost Algerian margin and basin. We performed a tomographic inversion of traveltimes along a 100-km-long wide-angle seismic profile shot over 40 ocean bottom seismometers offshore Mostaganem (Northwestern Algeria). The resulting velocity model and multichannel seismic reflection profiles show a thin (3–4 km thick) oceanic crust. The narrow ocean–continent transition (less than 10 km wide) is bounded by vertical faults and surmounted by a narrow almost continuous basin filled with Miocene to Quaternary sediments. This fault system, as well as the faults organized in a negative-flower structure on the continent side, marks a major strike-slip fault system. The extremely sharp variation of the Moho depth (up to 45 ± 3°) beneath the continental border underscores the absence of continental extension in this area. All these features support the hypothesis that this part of the margin from Oran to Tenes, trending N65–N70°E, is a fossil subduction-transform edge propagator fault, vestige of the propagation of the edge of the Gibraltar subduction zone during the westward migration of the Alborán domain.