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: Surface nuclear magnetic resonance (surface-NMR) is a promising technique for exploring shallow subsurface aquifer structures. Surface-NMR can be applied in environments that are characterized as a 1-D layered Earth. The technique utilizes a single loop and is referred to as magnetic resonance sounding. The technique referred to as magnetic resonance tomography (MRT) allows complex 2-D aquifer structures to be explored. Currently, MRT requires multiple loops and a roll along measurement scheme, which causes long survey time. We propose a loop layout using an elongated transmitter and an in-loop receiver arrays (ETRA) to conduct a 2-D survey with just one measurement. We present a comprehensive comparison between the new layout and the common approaches based on sensitivity and resolution analyses and show synthetic and field data. The results show that ETRA generates subsurface images at sufficient resolution with significantly lower survey times than other loop layouts.

Description: We studied ionospheric responses to the 2012 April 11 M w 8.6 North Sumatra earthquake using total electron content (TEC) measurements with the regional Global Navigation Satellite System network. This earthquake ruptured the oceanic lithosphere off the Indian Ocean coast of North Sumatra, and is known as the largest strike-slip earthquake ever recorded. Coseismic ionospheric disturbances (CIDs) with rapid TEC enhancement of a few TEC units propagated northward with a speed of acoustic waves (~1 km s –1 ). Resonant atmospheric oscillation with a frequency ~4 mHz have been found as monochromatic oscillation of TEC lasting for an hour after the main shock and the largest aftershock. We compared CID amplitudes of 21 earthquakes world-wide with moment magnitudes ( M w ) 6.6–9.2. They roughly obeyed a law such that CID amplitude increases by two orders of magnitude for the M w increase of three. The 2012 North Sumatra earthquakes slightly deviated negatively from the trend possibly reflecting their strike-slip mechanisms, that is small vertical crustal movements for their magnitudes.

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: Broad-band magnetic susceptibility (MS) measurement, a novel magnetic method capable of quantifying a narrow grain size distribution (GSD) of superparamagnetic (SP) particles by measuring low-field MS at a number of frequency steps spanning four orders of magnitude, has been tested in a loess/palaeosol section at Luochuan in the Chinese Loess Plateau. The studied succession consists of sequences from the latest palaeosol unit (S0) to the upper part of the loess unit (L2), spanning the last glacial–interglacial cycle. Reconstructed GSDs consist of volume fractions on the order of 10 –24 m 3 , and the mean GSDs are modal but with distinctive skewness among the loess, the weakly developed palaeosols (weak palaeosols), and the mature palaeosols. This indicates that the mean volume of SP particles in this loess/palaeosol sequence tends to increase during the transition from loess -〉 weak palaeosol -〉 palaeosol, an indication of grain growth as pedogenesis progresses. Total frequency dependence, or TFD(per cent), the difference between 130 at the lowest (130 Hz) and 500k at the highest (500 kHz) frequencies normalized to 130 , is judged to be a more suitable index than previous frequency dependence parameters for the concentration of SP particles. TFD(per cent) has a strong correlation with 130 , showing a continuous ‘growth curve’ with the rate of increase being highest for the loess, moderate for the weak palaeosols, and saturated for the palaeosols. The characteristic curve suggests that smaller SP particles are preferentially formed in the earlier stage of pedogenesis rather than the later phase when even larger particles are formed in mature palaeosols. These results demonstrate that the broad-band MS measurement method will be useful for the quantitative assessment of magnetic nanoparticles in soils and sediments.

Description: Environmental magnetism can provide simple and fast methods to semi-quantify the contamination levels by heavy metals in urban areas from the relationships between the magnetic properties and the heavy metal concentrations. The aim of this study is to explore the interpretation of selected magnetic parameters as proxies for the evaluation of heavy metal contamination in urban dusts. Dust samples were collected from different districts of Nanjing, Southeast China. The magnetic properties of dusts were analysed and heavy metal (Fe, Al, As, Ba, Cd, Cr, Co, Cu, Hg, Mn, Mo, Ni, Pb, Sr, V and Zn) concentrations were measured. Magnetic minerals, mainly from anthropogenic activities, are dominated by coarse stable single domain-grained ferrimagnetic minerals. The – T curves indicated that the main magnetic minerals are magnetite and hematite. Magnetic properties exhibited similar spatial distribution and significant positive correlations with the concentrations of most metals and with the pollution load index. Magnetic properties had a strong link with the abundance of heavy metals derived from industrial emission and traffic activities, whereas there was a poor correlation with the concentrations of metals from multi-sources such as commercial/domestic sources and natural processes. Besides the low-frequency magnetic susceptibility ( LF ) and the saturation isothermal remanent magnetization, other magnetic parameters can be considered as efficient indicators in the assessment of heavy metal contamination. The linkage between the magnetic properties and heavy metal concentrations in street dusts depends on the sources and nature of their magnetic fraction. Environmental magnetism analyses may provide simple and rapid methods for the assessment of heavy metal contamination in urban street dusts.

Description: We present in this paper an alternative approach to modelling and inversion of surface nuclear magnetic resonance (sNMR) data that has numerous attractive characteristics. By considering the forward and inverse problem in the frequency-domain (FD) it is possible to heavily truncate the band-limited data set with no appreciable loss of information. Furthermore, it introduces a natural method of signal demodulating and sidesteps the rotating frame transformation, which can be difficult to apply to large volumes. By instead remaining in the laboratory frame, phase shifts due to off-resonance transmission and static variation in the Larmor frequencies become simpler to account for. For these reasons, we present the first practical scheme for comprehensively inverting the complex sNMR data set. The use of complex data allows for improved depth and decay constant resolution. However, it requires the knowledge of subsurface electrical conductivity, and by corollary the ability to accurately model those effects in the sNMR record. Finally, the complex FD signal is sensitive to dephasing of the sNMR record due to static magnetic field inhomogeneity. We illustrate the ability to detect dephasing in synthetic and field data and demonstrate an effective inversion which is able to solve for static dephasing effects. This results in better estimation of meaningful decay constants as well as reduced error in extrapolated initial signal amplitude. Both of these parameters are critical for using sNMR in applications requiring estimation of hydraulic parameters.

Description: We report a palaeomagnetic investigation of the last full geomagnetic field reversal, the Matuyama-Brunhes (M-B) transition, as preserved in a continuous sequence of exposed lacustrine sediments in the Apennines of Central Italy. The palaeomagnetic record provides the most direct evidence for the tempo of transitional field behaviour yet obtained for the M-B transition. 40 Ar/ 39 Ar dating of tephra layers bracketing the M-B transition provides high-accuracy age constraints and indicates a mean sediment accumulation rate of about 0.2 mm yr –1 during the transition. Two relative palaeointensity (RPI) minima are present in the M-B transition. During the terminus of the upper RPI minimum, a directional change of about 180 ° occurred at an extremely fast rate, estimated to be less than 2 ° per year, with no intermediate virtual geomagnetic poles (VGPs) documented during the transit from the southern to northern hemisphere. Thus, the entry into the Brunhes Normal Chron as represented by the palaeomagnetic directions and VGPs developed in a time interval comparable to the duration of an average human life, which is an order of magnitude more rapid than suggested by current models. The reported investigation therefore provides high-resolution integrated palaeomagnetic and radioisotopic data that document the fine details of the anatomy and tempo of the M-B transition in Central Italy that in turn are crucial for a better understanding of Earth's magnetic field, and for the development of more sophisticated models that are able to describe its global structure and behaviour.

Description: The anisotropy of magnetic susceptibility (AMS) of soft rocks was measured in order to distinguish between the effect of remote and local strain fields, determine the size of the related inelastic damage zone and resolve the fault-plane solutions of past earthquakes. The AMS fabrics were explored next to late Pleistocene syndepositional normal faults (total displacement up to ~3.5 m) that cross soft lacustrine rocks within the seismically active Dead Sea basin. ‘Deposition fabrics’ prevail meters away from the fault planes and are characterized by scattered maximum and intermediate principal AMS axes. ‘Deformation fabrics’ are detected up to tens of centimetres from the fault planes and are characterized by well-grouped AMS axes, in which one of the principal axes is parallel to the strike of the nearby fault. Variations in the AMS fabrics and magnetic lineations define the size of the inelastic damage zone around the faults. The results demonstrate that the deformation-driven magnetic fabrics and the associated inelastic damage zones are compatible with coseismic dynamic faulting and the effects of the local strain field during earthquakes. Most of the AMS fabrics show a conspicuous similarity to that of the fault-plane solutions, i.e., the principal AMS axes and instantaneous strain ellipsoids are coaxial. These results suggest a novel application of the AMS method for defining the shape and size of the damage zones surrounding dynamic faults and determining the full tensor of the local strain field.

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: Very low frequency (VLF) electromagnetic waves that penetrate conductive magma-filled dykes generate secondary fields on the surface that can be used to invert for dyke properties. The model used for the interpretation calculates currents induced in a conductive strip by an inducing field that decays exponentially with depth due to the conductivity of the surrounding medium. The differential equations are integrated to give an inhomogeneous Fredholm equation of the second kind with a kernel consisting of a modified Bessel function of the second kind. Numerical methods are typically used to solve for the induced currents in the strip. In this paper, we apply a modified Galerkin–Chebyshev method, which involves separating the kernel into source and field spectra and integrating the source terms to obtain a matrix equation for the unknown coefficients. The incident wave is expressed as a Chebyshev series. The modified Bessel function is separated into a logarithmic singularity and a non-singular remainder, both of which are expanded in complex Chebyshev polynomials. The Chebyshev coefficients for the remainder are evaluated using a fast Fourier transform, while the logarithmic term and incident field have analytic series. The deconvolution then involves a matrix inversion. The results depend on the ratio of strip-size to skin-depth. For infinite skin-depth and a singular conductivity distribution given by $\tau _0 a/\sqrt{a^2 - z^2 }$ (where 0 is the conductance, a is the half-length and z the distance from the centre), Parker gives an analytic solution. We present a similar analytic series solution for the finite skin-depth case, where the size to skin depth ratio is small. Results are presented for different ratios of size to skin depth that can be compared with numerical solutions. We compare full-space and half-space solutions. A fit of the model to VLF data taken above a magma filled dykes in Hawaii and Mt Etna demonstrates that while properties such as depth to top, conductivity ratio, tilt and dip can be determined, the depth to bottom is indeterminate due to the exponential decay of the inducing field.

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: Correlated data errors (noise) are common in magnetotelluric (MT) data, but MT inversions typically neglect error correlations without investigating the impact of this simplification on inversion results. This paper examines effects of neglecting frequency- and spatially correlated noise on MT inversion, based on a nonlinear Bayesian formulation which quantifies the uncertainties of inversion results in terms of marginal posterior probability densities and credibility intervals. To do so, data with frequency- and spatially correlated noise of differing degrees are generated for several layered (1-D) synthetic cases. Bayesian MT inversions are carried out for these data sets with and without accounting for error correlation (i.e. applying full and diagonal covariance matrices, respectively, in the inversion), and the results are compared. For cases with noise that is strongly correlated over frequency or space, parameter uncertainties estimated using the diagonal-covariance simplification (neglecting error correlations) are found to often be significantly underestimated compared with results computed using the full covariance matrix.

Description: We report a new method to infer continuous time-series of the declination, inclination and intensity of the magnetic field from archaeomagnetic data. Adopting a Bayesian perspective, we need to specify a priori knowledge about the time evolution of the magnetic field. It consists in a time correlation function that we choose to be compatible with present knowledge about the geomagnetic time spectra. The results are presented as distributions of possible values for the declination, inclination or intensity. We find that the methodology can be adapted to account for the age uncertainties of archaeological artefacts and we use Markov chain Monte Carlo to explore the possible dates of observations. We apply the method to intensity data sets from Mari, Syria and to intensity and directional data sets from Paris, France. Our reconstructions display more rapid variations than previous studies and we find that the possible values of geomagnetic field elements are not necessarily normally distributed. Another output of the model is better age estimates of archaeological artefacts.

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: We present a rigorous method for interpolation of electric and magnetic fields close to an interface with a conductivity contrast. The method takes into account not only a well-known discontinuity in the normal electric field, but also discontinuity in all the normal derivatives of electric and magnetic tangential fields. The proposed method is applied to marine 3-D controlled-source electromagnetic modelling (CSEM) where sources and receivers are located close to the seafloor separating conductive seawater and resistive formation. For the finite-difference scheme based on the Yee grid, the new interpolation is demonstrated to be much more accurate than alternative methods (interpolation using nodes on one side of the interface or interpolation using nodes on both sides, but ignoring the derivative jumps). The rigorous interpolation can handle arbitrary orientation of interface with respect to the grid, which is demonstrated on a marine CSEM example with a dipping seafloor. The interpolation coefficients are computed by minimizing a misfit between values at the nearest nodes and linear expansions of the continuous field components in the coordinate system aligned with the interface. The proposed interpolation operators can handle either uniform or non-uniform grids and can be applied to interpolation for both sources and receivers.

Description: According to field observations, electromagnetic (EM) signals accompanying seismic waves can be recorded. The orders of magnitude of observed coseismic electric and magnetic signals associated with earthquakes are usually around 1 to 10 1 mV km –1 and 10 –2 to 1 nT, respectively. In this paper, we carry out numerical simulation of coseismic EM signals associated with seismic waves due to electrokinetic effect and compare with field observations. The seismic source is represented by a finite fault measuring 15 x 9 km 2 with a max slip displacement 1.5 m, corresponding to a M w 5.9 earthquake. While using the EM surface boundary condition of continuous horizontal EM components, the magnetic signals only accompany the late-arriving S waves at receiver near the ground surface. This is obviously different from field observations. Thus, we adopt another EM surface boundary condition which assumes the ground surface carries surface charge. For the used half-space model, a surface-charge density magnitude | Q sc | in excess of 10 –4 C m –2 is sufficient to make horizontal magnetic components clearly show up at the whole time duration of seismic waves. When | Q sc | increases, the contribution of surface-charge density to coseismic EM signals becomes more and more dominant comparing with that of the seismically induced streaming-current. We estimate the Q sc expected at the Earth's surface might be a value between –5 x 10 –4 and –0.1 C m –2 by the comparison between numerical results and field observations. The vertical magnetic signals only accompany the late-arriving seismic waves, because they are theoretically only induced by SH wave. The field observation results of vertical magnetic signals may be resulted from the scattering effect or the seismic dynamo effect. We conclude that electrokinetic effect combined with surface-charge assumption is one possible generation mechanism of the observed coseismic EM signals.

Description: Fifteen sites of lava flows from Martinique Island (FWI) have been selected to document the geomagnetic field in the Caribbean area over the past 2.5 Ma and further constrain the time-averaged field during this period. Identical characteristic directions were isolated using both AF and thermal stepwise demagnetization techniques in all flows. Nine mean-site directions have a normal polarity, while three others are reversed. The mean geomagnetic pole position obtained after reducing all directions to the same polarity is indistinguishable from the present north geographic pole. The dispersion is at least 8° larger than the values derived from the time-averaged field models and remains unexplained otherwise than resulting from the relatively small number of directions. The other three flows are characterized by large deviations from the expected north–south direction. One lava flow dated at 1.69 Ma (±0.02 Ma) is likely associated with a transitional field during the Gilsà subchron. The lava flow dated at 770 ka (±11 ka) coincides with the age of the Brunhes-Matuyama geomagnetic reversal and is also coeval with another intermediate flow of the same age found at Guadeloupe Island. The 617 ka (±52 ka) old unit is characterized by reversed directions that are evidently not related to the last reversal, but with other reversed polarity and transitional lava flows of the same age recorded, respectively at Mexico and La Palma island. We infer that the presence of reversed directions with the same age at distinct localities confirms that a short episode of reversed polarity has occurred during this period.

Description: Presently, most loess/palaeosol magnetic susceptibility records are interpreted as following either the wind-vigour model or the pedogenic enhancement model. However redoxomorphic processes induced by waterlogging, often referred to gleying in the loess literature, are also known to alter loess deposits but their impact on loess/palaeosol magnetic susceptibility records has received little attention. The reported rock magnetic study aims to characterize the mineral magnetic response of loess to waterlogging-induced redoxomorphic processes, thus improving our understanding of mineral magnetic changes within loess deposits with respect to environmental and climate conditions. The Nussloch loess-palaeosol deposit (Rhine Valley, Germany) was targeted because it is one of the best-studied Pleniglacial deposits for Western Europe in which numerous tundra gley intervals have been identified. Moreover, a comprehensive high-resolution environmental magnetism study has never been undertaken for this site. Various rock magnetism experiments were conducted at both room and low temperatures to characterise the composition, concentration and relative magnetic grain size of the mineral magnetic assemblage. The relative changes in magnetic parameters within the investigated loess interval are primarily controlled by (1) varying concentrations of coarse-grained ferrimagnetic particles of detrital (aeolian) origin and (2) dissolution of fine-grained ferrimagnetic particles related to in situ post-depositional alteration promoted by waterlogging-induced redoxomorphic processes. Goethite is found to be ubiquitous throughout the studied interval and is argued to have both a primary (aeolian) and secondary ( in situ ) origin. We conclude, that redoxomorphic processes induced by waterlogging, if present, will hinder the interpretation of magnetic susceptibility variations within loess and palaeosol deposits following the expected relationships dictated by the wind-vigour and the pedogenic enhancement magnetism models.

Description: Palaeosecular variation (PSV), as estimated from the scatter of remanent magnetization directions or poles, can be used to shed light to processes in the geodynamo, and potentially, to model the growth of the solid inner core. To understand the temporal aspects of the geomagnetic field behavior in the far past, we have calculated the scatter of palaeomagnetic poles (S) from a set of 55 high-quality observations derived from a new Precambrian paleomagnetic data compilation. Our analysis supports former Phanerozoic and Precambrian analyses of PSV, which favour a lower reversal rate, a higher stability of the geodynamo and a substantially smaller size of the inner core prior to 1.5 billion years ago.

Description: A palaeomagnetic study has been carried out in 29 cores drilled at six different sites from the volcanic products of Lomo Negro eruption (El Hierro, Canary Islands, Spain). Systematic thermal and alternating field demagnetization of the samples’ natural remanent magnetization revealed a northward, stable palaeomagnetic direction similar in all the samples. Rock magnetic experiments indicate that this palaeomagnetic component is carried by a mixture of high-Ti and low-Ti titanomagnetite crystals typical of basaltic lithologies that have experienced a significant degree of oxyexsolution during subaerial cooling. The well constrained palaeomagnetic direction of Lomo Negro lavas was used to perform a palaeomagnetic dating of the volcanic event, using the SHA.DIF.14k global geomagnetic model restricted for the last 3000 yr. It can be unambiguously concluded that Lomo Negro eruption occurred well before the previously proposed date of 1793 AD, with three different age ranges being statistically possible during the last 3 ka: 115 BC-7 AD, 410–626 AD and 1499–1602 AD. The calibration of a previously published non-calibrated 14 C dating suggests a XVI c. date for Lomo Negro eruption. This conclusion leaves open the possibility that the seismic crisis occurred at El Hierro in 1793 AD was related to an intrusive magmatic event that either did not reach the surface or either culminated in an unregistered submarine eruption similar to the one occurred in 2011–2012 at the southern off-shore ridge of the island.

Description: We have performed an extended palaeomagnetic study of the Oslo Graben volcanics, compared to the study of half a century ago by van Everdingen, using modern techniques and a four times larger amount of sites, plus additional rock magnetic experiments. We conclude that the average direction ( D = 204.0, I = –37.9, k = 46.9, α 95 = 2.0) and associated palaeomagnetic pole ( = 48.3, = 155.5, K = 52.2, A 95 = 1.9) of the Krokskogen and Vestfold volcanics together are statistically identical to those of the earlier study. This gives confidence in the fact that older palaeomagnetic studies can be reliable and robust, even though methods have improved. Our larger number of samples, and better age constraints, enable us to separate the data into two major intervals: the younger, on average, Krokskogen area and the older Vestfold area. The results show firstly that palaeolatitudes are slightly higher than predicted by the latest apparent polar wander path (APWP) for Eurasia by Torsvik et al. These data support an early Permian Pangaea A configuration and do not necessitate a Pangaea B configuration. The larger data set also allows us to assess the distribution of the characteristic remanent magnetization directions of the Oslo Graben in terms of geomagnetic field behaviour, which were acquired during a long period of dominantly single polarity the Permo-Carboniferous Reversed Superchron (PCRS). The distributions show a significantly lower virtual geomagnetic pole (VGP) scatter at the observed (low) latitudes than expected from a compilation from lavas of the last 5 Myr. The data do however show excellent agreement with the scatter observed both during the Cretaceous Normal Superchron and the PCRS. A comparison of the directional distributions in terms of elongation is less discriminating, since the large errors in all cases allow a fit to the predicted elongation/inclination behaviour of the TK03.GAD model.

Description: We illustrate a new imaging method to estimate the depth to the sources of potential fields and the structural index. The method consists of applying the Depth from EXtreme Point (DEXP) transformation to the ratio (R) between two different-order partial derivatives of the field. While the scaling function of the potential field depends on the structural index, we show that the scaling function of R merely depends on the difference between the two used orders of differentiation. This allows three main features to be established for the DEXP transformation of R: (1) it is independent from the structural index; (2) the estimation of the source depths is fully automatic, simply consisting in the search of position of the extreme points of the DEXP image and (3) the structural index of each source is finally determined from the scaling function or the extreme points using the estimated depth. Besides the well-known characteristics of the DEXP transformation, such as high-resolution and stability, the DEXP transformation of R enjoys one more relevant feature: it can be applied to multisource cases, yielding simultaneously correct estimations of structural index and depth for each source in the same image. However, while the DEXP transformation is a linear transformation of the field, the DEXP transformation of R is non-linear, and a procedure is described to circumvent the non-linear effects. The method is tested with synthetic examples and the estimated source parameters show a good agreement with the true values. The method was applied also to real magnetic data from the Pima copper mine, Arizona, USA, Hamrawien area, Egypt and Cataldere, Bala district of Turkey. The results are consistent with the known information about the causative sources.

Description: Variational data assimilation (4DVar) is a powerful technique for tuning dynamic models to observations, in order not only to forecast future time evolution of the system, but to make inferences about quantities that are otherwise unconstrained by observation. We apply this technique, well-grounded in meteorology and oceanography, to the Earth's core where incompressible fluid motions in an electrically conducting medium are responsible for magnetic field generation. Our dynamic model's momentum equation neglects inertia such that the entire evolution depends only on the structure of the initial magnetic field; time evolution of the system is solely governed by the equation of magnetic induction. Nevertheless the dynamic system encompasses the effects of rotation, Lorentz forces and viscosity and aims to mimic a reasonable force-balance in the Earth's core. Building on the work of Li et al. , in order to optimize the data-fit subject to the dynamics, we further develop the mathematical structure of the adjoint equations of the system. We address the feasibility of recovering 3-D spatial properties of the system using only time-varying 2-D observations of different character. Using closed-loop testing, we demonstrate the retrievability of the initial state (and thus the entire trajectory) of the system over convective timescales, when sampling in regions in which magnetic induction dominates over diffusion. The results suggest the possibility of retrieving the entire trajectory of the dynamo system of the Earth using the 400-yr model of secular variation gufm1 .

Description: We investigate polarity reversals in the geodynamo using a rotating, convection-driven dynamo model. As the flow in rapidly rotating convection is dominated by columns aligned with the axis of rotation, the focus is on the dynamics of columnar vortices. By studying the growth of a seed magnetic field to a stable axial dipole field, we show that the magnetic field acts in ways that significantly enhance the relative helicity between cyclonic and anticyclonic vortices. This flow asymmetry is the hallmark of a dipolar dynamo. Strong buoyancy, on the other hand, offsets the effect of the magnetic field, establishing parity between positive and negative vortices. As the dipole field is deprived of the helicity required to support itself, the dynamo is pushed into a reversing state. This is a likely regime for polarity reversals in the Earth's core. The integral lengthscale at which buoyancy injects energy is not significantly different from the convective flow lengthscale, which implies that buoyancy does not feed vortices at the small scales where non-linear inertia is present. The lengthscale at which the Lorentz force acts in the reversing dynamo is small, which may allow the passive presence of non-linear inertia in the small scales.

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: A new methodology to estimate magnetotelluric (MT) tensor relationships, called Estimation of Local transfer-functIons by Combining Interstation Transfer-functions (ELICIT), is proposed whereby the MT tensor relationships of the local site are derived using only interstation transfer functions. The MT impedance tensor and the geomagnetic transfer function at the local site are characterised by combining interstation tensor relationships between electric and magnetic fields at the local site with the horizontal magnetic fields acquired at a neighbouring site. The main property of the proposed method is that the employed interstation transfer functions are independently constrained, without the need to acquire the electric and the magnetic fields at the local site simultaneously to recover the local MT tensor relationships. Due to this property, the ELICIT method offers new possibilities for MT data acquisition and processing, providing significant improvements when the magnetic time-series at the local site are affected by local noise or are truncated. Error analysis shows that, even when magnetic fields are truncated, the quality of the results obtained following the ELICIT method are similar to those we would obtain if the magnetic fields had not been truncated. Another important property is that different neighbouring sites can be used to recover the tensor relationships at the local site. Averaging of results obtained using different neighbouring sites can be performed to improve the statistics. For our example data, when the ELICIT method is used to improve the statistics, errors of the estimates for periods between 1000 and 20 000 s periodicities are clearly reduced. All interstation transfer functions are calculated doing remote reference and the bootstrap method is used to compute the errors, when necessary. Long period magnetotelluric data acquired in the Pyrenees and in the Atlas Mountains in Morocco, and magnetic data provided by Fürstenfeldbruck magnetic observatory have been used to test the proposed ELICIT method, with positive results. Due to the lack of requirement that the electric and the magnetic fields of the local site be acquired simultaneously, the proposed method also offers new possibilities for MT data acquisition, optimizing the available instrumentation.

Description: The robust statistical model of a Gaussian core contaminated by outlying data that underlies robust estimation of the magnetotelluric (MT) response function has been re-examined. The residuals from robust estimators are systematically long tailed compared to a distribution based on the Gaussian, and hence are inconsistent with the robust model. Instead, MT data are pervasively described by the alpha stable distribution family whose variance and sometimes mean are undefined. A maximum likelihood estimator (MLE) that exploits the stable nature of MT data is formulated, and its two-stage implementation in which stable parameters are first fit to the data and then the MT responses are solved for is described. The MLE is shown to be inherently robust, but differs from the conventional robust estimator because it is based on a model derived from the data, while robust estimators are ad hoc , being based on the robust model that is inconsistent with actual data. Propriety versus impropriety of the complex MT response was investigated, and a likelihood ratio test for propriety and its null distribution was established. The Cramér-Rao lower bounds for the covariance matrix of proper and improper MT responses were specified. The MLE was applied to exemplar long period and broad-band data sets from South Africa. Both are shown to be significantly stably distributed using the Kolmogorov–Smirnov goodness of fit and Ansari-Bradley non-parametric dispersion tests. Impropriety of the MT responses at both sites is pervasive, hence the improper Cramér-Rao bound was used to estimate the MLE covariance. The MLE is shown to be nearly unbiased and well described by a Gaussian distribution based on bootstrap simulation. The MLE was compared to a conventional robust estimator, establishing that the standard errors of the former are systematically smaller than for the latter and that the standardized differences between them exhibit excursions that are both too frequent and too large to be described by a Gaussian model. This is ascribed to pervasive bias of the robust estimator that is to some degree obscured by their systematically large confidence bounds. Finally, a series of topics for further investigation is proposed.

Description: Broad-band and long period magnetotelluric (MT) data were acquired at 39 stations along five NNW-SSE profiles crossing the Iapetus Suture Zone (ISZ) in Ireland. Regional strike analyses indicate that the vast majority of the MT data is consistent with an assumption of a 2-D geo-electric strike direction. Strike is N52°E for the three easternmost profiles and N75°E for the two westernmost profiles; these directions correlate well with the observed predominant geological strike of the study region. 2-D inversions of the galvanic distortion-corrected TE and TM mode data from each profile are shown and discussed. As mapped geological variations between the neighbouring profiles suggest a heterogeneous subsurface, it is important to verify the robustness of the presence and geometries of prominent conductivity anomalies by employing 3-D forward and inverse modelling. A high conductivity layer (resistivity of 1–10 m), found at middle to lower crustal depths and presumed to be indicative of metamorphosed graphitic sediments rich in sulphides deposited during the convergence of the Laurentian and Avalonian continents, essentially constitutes the electrical signature of the ISZ. Shallow conductors observed are probably due to black shales that were widely deposited within the sedimentary accretionary wedge during Ordovician time. We interpret the moderately low resistivity at shallow depths from west to east across Ireland as indicative of an increase in maturity of the black shales in the easterly direction. From our conductivity models the southern extent of the ISZ is inferred to lie between the Navan Silvermines Fault and the Navan Tipperary Line, and shows clear resistivity contrast along all the profiles at the southern MT stations. The change in resistivity deduced from the 2-D models is spatially related to the composition of Lower Palaeozoic Ordovician, Silurian, Devonian and Carboniferous rocks. At upper mantle depths of about 60 km, a high conductivity block below the central MT stations is found to lie within the accretionary wedge of the Iapetus suture, and the location of the conductive anomaly corroborates reasonably well with the inferred spreading head of the putative Iceland plume-related magmatic intrusion. The low resistivity upper crust beneath the ISZ is indeed rich in Ordovician rocks with black shale content in the eastern as well as the central part; the western part is largely underlain by a highly resistive block of volcanic and metamorphosed rocks forming crystalline basement.

Description: This paper presents new directional archeomagnetic data from nine Meso-/Neolithic fireplaces, sampled in a cave shelter, at Arconciel, in western Switzerland. Rock magnetic measurements indicate a homogenous magnetic mineralogy in all fireplaces, with magnetite as the main magnetic carrier. The remanent magnetization is stable and generally shows one characteristic directional component. Nine new directions, which were obtained from Arconciel, are combined with 356 other archeomagnetic data from a circular area with a radius of 700 km around this site, to obtain a penalized least square spline fit for the past 9000 yr. We found in general good agreement with other local compilations, such as the Balkan curve, the regional SCHA.DIF.8k model and with lake sediments from UK, Fennoscandia and Switzerland. Nevertheless, a time lag of several centuries is observed for a declination maximum between the archeomagnetic spline fit and the other European data records around 5900 BC. This time lag is also observed in the Swiss lake sediment record; therefore we interpret this shift as a local feature of the Earth's magnetic field.

Description: In Christian religion the sunrise is of great symbolic importance. Therefore, many churches constructed in the Middle Ages point towards geographic East. Although ‘easting’ of churches actually refers to the alignment towards the azimuth of sunrise on the individual churches’ patron's day, deviation of nave alignment from the geographic East direction is often assumed to be caused by the use of magnetic compasses. Therefore, the church alignment could provide information about historical magnetic declination. We investigate 124 churches in Lower Austria and 68 in northern Germany to clarify this question as well as the ‘easting’ hypothesis. Church orientations are determined from georeferenced satellite images. Metadata such as the construction year, possible reconstructions and the church patron are gathered to determine the date when current church direction was appointed, and to perform sunrise calculations. However, due to uncertainties of construction years and the declining importance of orientation tradition after the 15th century several churches are excluded from the study. Thus, 32 churches with reliable metadata remain for evaluation in each region. The analysis reveals a preferred alignment of naves towards geographic East in Lower Austria and northern Germany. The construction and alignment of churches was often affected by the pre-existing buildings and streets or topography and natural surroundings. Therefore, deviations from geographic East are more likely caused by town or landscape. The mean deviations from magnetic East in both regions are large compared to the mean deviations from geographic East and sunrise azimuths. Hence, the use of compasses cannot be confirmed. Despite a few churches indicating orientation according to their patron's day, a general trend cannot be observed in the data reviewed.

Description: A new finite difference solution to the global geomagnetic induction problem is developed and tested, based on a modified Lorenz gauge of the magnetic vector and electric scalar potentials and implementing a novel, overset ‘Yin-Yang’ grid that avoids unnecessary mesh refinement at the geographic poles. Previously used in whole-earth mantle convection models, the overset grid is built from a pair of partially overlapping mid-latitude latitude–longitude (lat/lon) grids, one of which is rotated with respect to the other for complete coverage of the sphere. Because of this symmetry, only one set of finite difference templates is required for global discretization of the governing Maxwell equations, a redundancy that is exploited for computational efficiency and multithreaded parallelization. Comparisons between solutions obtained by the proposed method show excellent agreement with those obtained by independent integral equation methods for 1-D, 2-D and 3-D problem geometries. The computational footprint of the method is minimized through a (non-symmetric) matrix-free BiCG-STAB iterative solver which computes finite difference matrix coefficients ‘on the fly’ as needed, rather than pulling stored values from memory. Scaling of the matrix-free BiCG-STAB algorithm with problem size shows behaviour similar to that seen with the (symmetric) QMR algorithm used in the Cartesian case from which the present algorithm is based. The proposed method may therefore provide a competitive addition to the existing body of global-scale geomagnetic induction modelling algorithms, allowing for resource-efficient forward modelling as the kernel for large-scale computing such as inversion of geomagnetic response functions, computational hypothesis testing and parametric studies of mantle geodynamics and physiochemical state.

Description: We present a robust and scalable solver for time-harmonic Maxwell's equations for problems with large conductivity contrasts, wide range of frequencies, stretched grids and locally refined meshes. The solver is part of the fully distributed adaptive 3-D electromagnetic modelling scheme which employs the finite element method and unstructured non-conforming hexahedral meshes for spatial discretization using the open-source software deal.II. We use the complex-valued electric field formulation and split it into two real-valued equations for which we utilize an optimal block-diagonal pre-conditioner. Application of this pre-conditioner requires the solution of two smaller real-valued symmetric problems. We solve them by using either a direct solver or the conjugate gradient method pre-conditioned with the recently introduced auxiliary space technique. The auxiliary space pre-conditioner reformulates the original problem in form of several simpler ones, which are then solved using highly efficient algebraic multigrid methods. In this paper, we consider the magnetotelluric case and verify our numerical scheme by using COMMEMI 3-D models. Afterwards, we run a series of numerical experiments and demonstrate that the solver converges in a small number of iterations for a wide frequency range and variable problem sizes. The number of iterations is independent of the problem size, but exhibits a mild dependency on frequency. To test the stability of the method on locally refined meshes, we have implemented a residual-based a posteriori error estimator and compared it with uniform mesh refinement for problems up to 200 million unknowns. We test the scalability of the most time consuming parts of our code and show that they fulfill the strong scaling assumption as long as each MPI process possesses enough degrees of freedom to alleviate communication overburden. Finally, we refer back to a direct solver-based pre-conditioner and analyse its complexity in time. The results show that for multiple right-hand sides the direct solver-based pre-conditioner can still be faster for problems of medium size. On the other hand, it also shows non-linear growth in memory, whereas the auxiliary space method increases only linearly.

Description: The configuration of the Pangea supercontinent has been a topic of intense debate for almost half a century, a controversy that stems from discrepancies between the geology-based Pangea-A and the palaeomagnetically based Pangea-B. Recent palaeomagnetic compilations aimed at resolving this controversy have identified the poor quality of palaeomagnetic data from Gondwana for Permian times as a major obstacle. Specifically, the vast majority of Gondwanan poles come from sedimentary rocks that are prone to biases from compaction or are poorly dated. Here, we present a new palaeomagnetic pole for cratonic South America based on impact melts from the 254.7 ± 2.5 Ma Araguainha impact structure (AIS). The granite basement, the impact-generated melt sheet and veins were sampled at 28 sites (169 specimens) and provided a reliable palaeomagnetic record similar to that of volcanic rocks. Alternating field and thermal demagnetization indicate a stable characteristic remanent magnetization carried by both magnetite and haematite. All sites but one show a single palaeomagnetic direction of normal polarity with a mean direction of Dec = 357.4°; Inc = –38.9°; N  = 28; k  = 62.35; α 95  = 3.5°, yielding a palaeomagnetic pole (AIS) at Lat = –84.2; Lon = 326.6; K  = 83.5; A 95  = 3.6°; S B  = 9.6°. The new pole provides a firm constraint on the position of Gondwana which is consistent with the Pangea A configuration.

Description: Reconstructions of the Holocene geomagnetic field and how it varies on millennial timescales are important for understanding processes in the core but may also be used to study long-term solar-terrestrial relationships and as relative dating tools for geological and archaeological archives. Here, we present a new family of spherical harmonic geomagnetic field models spanning the past 9000 yr based on magnetic field directions and intensity stored in archaeological artefacts, igneous rocks and sediment records. A new modelling strategy introduces alternative data treatments with a focus on extracting more information from sedimentary data. To reduce the influence of a few individual records all sedimentary data are resampled in 50-yr bins, which also means that more weight is given to archaeomagnetic data during the inversion. The sedimentary declination data are treated as relative values and adjusted iteratively based on prior information. Finally, an alternative way of treating the sediment data chronologies has enabled us to both assess the likely range of age uncertainties, often up to and possibly exceeding 500 yr and adjust the timescale of each record based on comparisons with predictions from a preliminary model. As a result of the data adjustments, power has been shifted from quadrupole and octupole to higher degrees compared with previous Holocene geomagnetic field models. We find evidence for dominantly westward drift of northern high latitude high intensity flux patches at the core mantle boundary for the last 4000 yr. The new models also show intermittent occurrence of reversed flux at the edge of or inside the inner core tangent cylinder, possibly originating from the equator.

Description: Several recent studies have used palaeomagnetic estimates of the virtual axial dipole moment to construct a quantitative stochastic model for fluctuations and reversals in the Earth's dipole field. We investigate the physical significance of the terms in a standard stochastic (Langevin) model using output from a numerical geodynamo model. The first term, known as the drift term, characterizes the slow adjustment of the dipole field toward a time-averaged state. We find that the timescale for this slow adjustment is set by the magnetic decay time of dipole fluctuations. These fluctuations are typically be represented by the first few decay modes. The second term is often called the noise term because it characterizes the influence of short-period convective fluctuations in the core. We establish a connection between the noise term and the rms variation in magnetic induction. Applying these results to the palaeomagnetic field suggests that the rms variation in dipole generation exceeds the mean rate of generation. Such large fluctuations may be necessary to permit magnetic reversals. Palaeomagnetic estimates of the drift term favour a high electrical conductivity in the core. A lower bound on electrical conductivity is 0.6 x 10 6 S m –1 . Similarly, we establish an upper bound on turbulent magnetic diffusivity (0.8 m 2 s –1 ), although realistic estimates may be much less.

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 demonstrate that first differences of polar orbiting satellite magnetic data in the along-track direction can be used to obtain high resolution models of the lithospheric field. Along-track differences approximate the north–south magnetic field gradients for non-polar latitudes. In a test case, using 2 yr of low altitude data from the CHAMP satellite, we show that use of along-track differences of vector field data results in an enhanced recovery of the small scale lithospheric field, compared to the use of the vector field data themselves. We show that the along-track technique performs especially well in the estimation of near zonal spherical harmonic coefficients. Moreover, lithospheric field models determined using along-track differences are found to be less sensitive to the presence of unmodelled external field contributions and problems associated with the polar gap are ameliorated. Experiments in modelling the Earth's lithospheric magnetic field with along-track differences are presented here as a proof of concept. We anticipate that use of such along-track differences in combination with east–west field differences, as are now provided by the Swarm satellite constellation, will be important in building the next generation of lithospheric field models.

Description: We develop an singular value decomposition-based compression of the Green's function matrix of an electromagnetic integral equation forward solver for global geomagnetic induction, on top of an fast Fourier transform reduction of the system to a block-diagonal form. With this approach, the memory usage and CPU time of Krylov subspace iterative solutions are significantly reduced at a very small cost of accuracy, making the accelerated forward solver well suited for 3-D inversions as well as forward simulations with multiple sources.

Description: The Fisher distribution is central to palaeomagnetism but presents several problems when used to characterize geomagnetic field directions as observed in sequences of volcanic rocks. First, it introduces a shallowing effect when used to define the mean of any group of directional unit vectors. This is problematic because it can suggest the presence of persistent non-axial dipole components when none are present. More importantly, it fails to capture the observed ‘long tail’ in distributions of both directions and associated virtual geomagnetic poles in terms of angular distance from a central direction. To achieve a good fit to data, it therefore requires the introduction of a second distribution (and therefore the estimation of additional parameters) or the arbitrary removal of data. Here we present a new distribution to describe palaeomagnetic directions and demonstrate that it overcomes both of these problems, generating robust indicators of both the central direction (or pole position) and the spread of palaeomagnetic data as defined by unit vectors. Starting from the assumption that poles (or directions) have an expected colatitude, rather than a mean location, we derive the spherical exponential distribution. We demonstrate that this new distribution provides a good fit to palaeomagnetic data sets from seven large igneous provinces between 15 and 65 Ma and also those produced by numerical dynamo models. We also use it to derive a new shape parameter which may be used as a diagnostic tool for testing goodness of fit of models to data and use this to argue for a shift in geomagnetic behaviour between 5 and 15 Ma. Furthermore, we point out that this new statistic can be used to determine the most appropriate distribution to be used when constructing confidence limits for poles.

Description: Haematite pigment is a common constituent of sedimentary rocks, but its contribution to the natural remanent magnetization of rocks is poorly understood. Here, we describe magnetic properties of two distinct pigment types that produce a characteristic decorative ‘print stone’ found in the ~2.5 Ga Mount McRae Shale Formation, Hamersley Province, Western Australia. Distinct magnetic remanence directions observed in the Print Stone can be correlated to each pigment type. By comparison with the Australian apparent polar wander path, the remanence carried by uniformly distributed pigment can be dated to ~15–25 Ma, while two age options, the Mesoproterozoic (~1.5 Ga) or the middle Carboniferous (~320–310 Ma), are permissible for the remanence carried by the pigment responsible for the distinctive ‘newsprint’ pattern. Magnetic properties and demagnetization characteristics of the different pigment types overlap significantly, and thus are not predictive of the dominant remanence carrier. Magnetic characteristics of the uniformly distributed pigment vary significantly on short spatial scales. Strong local control on pigment formation raises the possibility that a primary remanent magnetization may survive locally in pockets within sedimentary red bed formations.

Description: Remanent magnetizations of magnetites between single-domain (SD) threshold size (0.1 μm) and 20 μm have SD-like intensities and coercivities. This paper shows for the first time that magnetite's induced magnetization also has pseudo-single-domain behaviour. The first part of the paper reports temperature-dependent initial susceptibility data, k 0 ( T ), of sized magnetites and assesses their granulometric potential. The second part transforms coercive force data, H c ( T ), for the same magnetites into simulated k 0 ( T ) curves. The third part considers k 0 ( T ) results of coarse-grained mafic rocks as candidate sources of deep-seated magnetic anomalies. High-temperature susceptibility k 0 measured with a Kappabridge for eight fractions of crushed natural magnetites (median sizes of 0.6, 1, 3, 6, 9, 14, 110 and 135 μm) shows a progressive increase in the height of the Hopkinson peak below the Curie point as grain size decreases. The trend is systematic and has granulometric potential in the 1–14 μm range. Self-demagnetization should produce almost flat k 0 ( T ) in grains larger than SD size but experimentally, well-defined Hopkinson peaks are not limited to the finest grains. 1-μm magnetites have a peak 1.5 times k 0 at 20 °C and 14-μm grains have a peak of 1.25. Only 110 and 135 μm grains have T -independent k 0 . Using an empirical relationship between coercive force H c and k 0 , H c ( T ) data for the sized magnetites were used to simulate k 0 ( T ) results. A hump in the k 0 heating curve around 250 °C was traced to annealing out internal strains, evident in H c data measured in first heatings. For sizes ≤6 μm, observed Hopkinson peaks were smaller than predicted, possibly because of a previously unrecognized grain-size dependence of the empirical constant relating H c and k 0 . Two crystalline rocks, a gabbro and a diabase, combine SD-like Hopkinson peaks and multidomain (MD) flat ramps in their k 0 ( T ) data. In the diabase, a Hopkinson peak is prominent in separated plagioclase grains containing submicron magnetite, but is masked in whole-rock data. The gabbro has a clear superposition of SD and MD k 0 ( T ) functions in its whole-rock data, with a Hopkinson peak of 1.35. If oceanic layer-3 gabbros have similar susceptibility enhancement above 500 °C, they could be more important magnetic anomaly sources than room-temperature k 0 measurements on dredged or fault-uplifted samples would suggest.

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: We investigate the influence of different thermal and velocity boundary conditions on numerical geodynamo models. We concentrate on the implications for magnetic field morphology, heat transport scaling laws, force balances and generation mechanisms. The field morphology most strongly depends on the local Rossby number, but there is some variation in the dipolarity of the field with boundary condition. Scaling laws also depend on the boundary conditions, but a diffusivity-free scaling is a good first order approximation for all our dipolar models. Our multipolar models, however, obey different scaling laws from dipolar models implying a different force balance in these models. We find that our dipolar models have a stronger degree of Lorentz–Coriolis balance compared to our multipolar models which have a stronger degree of Lorentz-inertial balance.The models with a stronger Lorentz–Coriolis dominance can be generated by either α, α 2 or α 2 mechanisms whereas the models with a stronger Lorentz-inertial balance are all α 2 dynamos. These results imply that some caution is necessary when extrapolating results from dynamo models to Earth-like parameters since the choice of boundary conditions can have important effects.

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: Lake sediments are excellent sources of palaeoenvironmental and palaeoclimatic information because they provide continuous and high-resolution records. South America is of particular interest because it is the only landmass that stretches southward into the Pacific and Atlantic Oceans towards Antarctica. The aim of this study is to explore the relationship of magnetic parameters with elemental and palaeobiological data of Laguna Potrok Aike to develop a model of lake-level changes and related hydrological and climatic fluctuations. Magnetic measurements were performed on subsamples from 15 500 cal. BP to the present, and associated rock magnetic parameters were calculated to infer magnetic mineralogy, concentration and grain size. According to the model, parameters dependent on magnetic concentration and grain size are directly related to lake-level changes. During dry periods, the remanent coercivity displays high values, whereas the proportion of magnetite is relatively low. Low percentages of greigite are observed, indicating that the water of the lake was stratified at least four times during the studied period, at approximately 10 300, 8900, 8500 and 8300 cal. BP. The preservation of greigite by inhibiting its complete transformation into pyrite is associated with a rapid burial that occurs with high sedimentation rates. Thermal stratification could be caused by a slight cooling in the area triggered by a weakening of the Westerlies and/or low activity of the sun, sum to the effect of a flood of melt water in the North Atlantic.

Description: Measurement of magnetic vector or tensor quantities, namely of field or field gradient, delivers more details of the underlying geological setting in geomagnetic prospection than a scalar measurement of a single component or of the scalar total magnetic intensity. Currently, highest measurement resolutions are achievable with superconducting quantum interference device (SQUID)-based systems. Due to technological limitations, it is necessary to suppress the parasitic magnetic field response from the SQUID gradiometer signals, which are a superposition of one tensor component and all three orthogonal magnetic field components. This in turn requires an accurate estimation of the local magnetic field. Such a measurement can itself be achieved via three additional orthogonal SQUID reference magnetometers. It is the calibration of such a SQUID reference vector magnetometer system that is the subject of this paper. A number of vector magnetometer calibration methods are described in the literature. We present two methods that we have implemented and compared, for their suitability of rapid data processing and integration into a full tensor magnetic gradiometry, SQUID-based, system. We conclude that the calibration routines must necessarily model fabrication misalignments, field offset and scale factors, and include comparison with a reference magnetic field. In order to enable fast processing on site, the software must be able to function as a stand-alone toolbox.

Description: Motional induction in the ocean by tides has long been observed by both land and satellite measurements of magnetic fields. While these signals are weak (~10 nT) when compared to the main magnetic field, their persistent nature makes them important for consideration during geomagnetic field modelling. Previous studies have reported several discrepancies between observations and numerical predictions of the tidal magnetic signals and those studies were inconclusive of the source of the error. We address this issue by (1) analysing magnetometer data from ocean-bottom stations, where the low-noise and high-signal environment is most suitable for detecting the weak tidal magnetic signals, (2) by numerically predicting the magnetic field with a spatial resolution that is 16 times higher than the previous studies and (3) by using four different models of upper-mantle conductivity. We use vector magnetic data from six ocean-bottom electromagnetic (OBEM) stations located in the Northwestern Pacific Ocean. The OBEM tidal amplitudes were derived using an iteratively re-weighted least-squares (IRLS) method and by limiting the analysis of lunar semidiurnal (M2), lunar elliptic semidinurnal (N2) and diurnal (O1) tidal modes to the night-time. Using a 3-D electromagnetic induction solver and the TPX07.2 tidal model, we predict the tidal magnetic signal. We use earth models with non-uniform oceans and four 1-D mantle sections underneath taken from Kuvshinov and Olsen, Shimizu et al. and Baba et al. to compare the effect of upper-mantle conductivity. We find that in general, the predictions and observations match within 10–70 per cent across all the stations for each of the tidal modes. The median normalized percent difference (NPD) between observed and predicted amplitudes for the tidal modes M2, N2 and O1 were 15 per cent, 47 per cent and 98 per cent, respectively, for all the stations and models. At the majority of stations, and for each of the tidal modes, the higher resolution (0.25° 0.25°) modelling gave amplitudes consistently closer to the observations than the lower resolution (1° 1°) modelling. The difference in lithospheric resistance east and west of the Izu–Bonin trench system seems to be affecting the model response and observations in the O1 tidal mode. This response is not seen in the M2 and N2 modes, thereby indicating that the O1 mode is more sensitive to lithospheric resistance.

Description: Magnetotelluric observations over one or 2-D structures that are distorted by 3-D electro-galvanic effects, need to be corrected for their proper conversion into reliable images of the subsurface electrical resistivity distribution. One of the most widely used approaches for correcting the data is the Groom–Bailey decomposition of the impedance tensor in terms of the unknown parameters of strike, twist and shear, along with the also unknown 2-D impedances. The standard approach for recovering the 2-D impedances is to solve numerically for all the unknowns as a non-linear inverse problem. In this work, we pose the recovery of the undistorted impedances in terms of a quadratic equation whose solutions filter out the distortions after a final tune for the appropriate shear parameter. The formula relies on two known invariants of the impedance tensor, the series and parallel invariants that have special immunity to some of the distorting parameters. Compared with the standard numerical recovery, the analytical solution provides more accurate results; they are clear-cut for both amplitudes and phases. The recovered amplitudes of the impedance are independent of strike, twist and shear, and the phases, in addition, of static effects. The recovery formula is transparent to random noise; hence the data preserve the original uncertainties. The applications to individual soundings of the COPROD2S1 data set and to a profile over a seismogenic region in Baja California, illustrate the effectiveness of the approach.

Description: Great progress has been made in the numerical simulation of planetary dynamos, though these numerical experiments still operate in a regime very far from the planets. For example, it seems unlikely that viscous forces are at all significant in planetary interiors, yet some of the simulations display a significant dependence on viscosity, and indeed in some of the simulations the dynamo mechanism is itself viscously driven, taking the form of helical Ekman pumping within columnar convection rolls. Given the similarity of the external magnetic fields observed in the terrestrial planets and gas giants, and the extremely small value of the Ekman number in all such cases, it seems natural to suppose that the underlying dynamo mechanism in these planets is simple, robust, independent of viscosity and insensitive to mechanical boundary conditions. A key step to identifying this mechanism is to determine the source of helicity in planetary cores, which itself should be robust, independent of viscosity and insensitive to boundary conditions. In this paper, we explore the possibility that the helicity in the core of the Earth arises from the spontaneous emission of inertial waves, driven by the equatorial heat flux in the outer core. We also ask if a similar mechanism might operate in other planets, and perhaps act to supplement the helicity driven by Ekman pumping in the (viscous) numerical simulations. We demonstrate that such waves do indeed produce the required helicity distribution outside the tangent cylinder. Moreover, we show that these waves inevitably propagate along the axis of the columnar vortices, and indeed they are the very mechanism by which the columnar vortices form in the first place and the means by which the columns subsequently evolve. We also calculate the emf induced by such axially propagating inertial waves and show that, in principle, this emf is sufficient to support a self-sustaining dynamo of the α 2 type. Finally, we derive the scaling laws for this kind of inertial-wave dynamo. We compare these predictions with the (imperfect) simulations, and also with what little we know about the Earth's core. The numerical experiments fall into two categories; the slowly rotating simulations which cannot sustain inertial waves at the small scales and the rapidly rotating (planet-like) ones which can. Our scaling laws are consistent with the latter class of simulations, and also with what we know about the Earth.

Description: The main sources of magnetic minerals in soils unaffected by anthropogenic pollution are iron oxides and hydroxides derived from parent materials through soil formation processes. Soil magnetic minerals can be used as indicators of environmental factors including soil forming processes, degree of pedogenesis, weathering processes and biological activities. In this study measurements of magnetic susceptibility are used to detect the presence and the concentration of soil magnetic minerals in topsoil and bulk samples in a small cultivated field, which forms a hydrological unit that can be considered to be representative of the rainfed agroecosystems of Mediterranean mountain environments. Additional magnetic studies such as isothermal remanent magnetization (IRM), anhysteretic remanent magnetization (ARM) and thermomagnetic measurements are used to identify and characterize the magnetic mineralogy of soil minerals. The objectives were to analyse the spatial variability of the magnetic parameters to assess whether topographic factors, soil redistribution processes, and soil properties such as soil texture, organic matter and carbonate contents analysed in this study, are related to the spatial distribution pattern of magnetic properties. The medians of mass specific magnetic susceptibility at low frequency ( lf ) were 36.0 and 31.1 10 –8 m 3 kg –1 in bulk and topsoil samples respectively. High correlation coefficients were found between the lf in topsoil and bulk core samples ( r  = 0.951, p  〈 0.01). In addition, volumetric magnetic susceptibility was measured in situ in the field ( is ) and values varied from 13.3 to 64.0 10 –5 SI. High correlation coefficients were found between lf in topsoil measured in the laboratory and volumetric magnetic susceptibility field measurements ( r  = 0.894, p  〈 0.01). The results obtained from magnetic studies such as IRM, ARM and thermomagnetic measurements show the presence of magnetite, which is the predominant magnetic carrier, and hematite. The predominance of superparamagnetic minerals in upper soil layers suggests enrichment in pedogenic minerals. The finer soil particles, the organic matter content and the magnetic susceptibility values are statistically correlated and their spatial variability is related to similar physical processes. Runoff redistributes soil components including magnetic minerals and exports fine particles out the field. This research contributed to further knowledge on the application of soil magnetic properties to derive useful information on soil processes in Mediterranean cultivated soils.

Description: State of the art numerical models of the Geodynamo are still performed in a parameter regime extremely remote from the values relevant to the physics of the Earth's core. In order to establish a connection between dynamo modelling and the geophysical motivation, it is necessary to use scaling laws. Such scaling laws establish the dependence of essential quantities (such as the magnetic field strength) on measured or controlled quantities. They allow for a direct confrontation of advanced models with geophysical constraints. We combine a numerical approach, based on a multiple linear regression method in the form of power laws, applied to a database of 102 direct numerical simulations (courtesy of U. Christensen), and a physical approach, based on energetics and forces balances. We show that previous empirical scaling laws for the magnetic field strength essentially reflect the statistical balance between energy production and dissipation for saturated dynamos. Such power based scaling laws are thus necessarily valid for any dynamo in statistical equilibrium and applicable to any numerical model, irrespectively of the dynamo mechanism. We show that direct numerical fits can provide contradictory results owing to biases in the parameters space covered in the numerics and to the role of a priori hypothesis on the fraction of ohmic dissipation. We introduce predictive scaling laws, that is relations involving input parameters of the governing equations only. We guide our reasoning on physical considerations. We show that our predictive scaling laws can properly describe the numerical database and reflect the dominant forces balance at work in these numerical simulations. We highlight the dependence of the magnetic field strength on the rotation rate. Finally, our results stress that available numerical models operate in a viscous dynamical regime, which is not relevant to the Earth's core.

Description: High-quality palaeomagnetic data for the early Carboniferous of Central Asia are scarce and the palaeogeographic evolution of this area prior to final amalgamation of the region east of the Ural mountains is still rather obscure. Here, we present palaeomagnetic data for early Carboniferous deposits from two areas in the Kyrgyz North Tianshan (NTS). Detailed rock-magnetic analysis indicates the presence of magnetite and haematite as magnetic carriers in these red sediments. In the Kazakh basin section (KEL), we identify a high-temperature component (HTC) of magnetization during stepwise thermal demagnetization at temperatures of up to ~680 °C yielding a site mean direction of D  = 176.2°, I  = –36.4°, k  = 57.4 and α 95  = 8.9° after tilt correction. Two HTCs of magnetization were identified in samples from the Sonkul Basin (DUN) with maximum blocking temperatures of ~600 °C (magnetite) and ~680 °C (haematite). The magnetite component was also identified with alternating field demagnetization. The resulting site mean directions for these two components identified in 16 and 14 sites, respectively, are D  = 149.3°, I  = –50.3°, k  = 73.6 and α 95  = 4.3° for the magnetite and D  = 139.6°, I  = –35.1°, k  = 71.6 and α 95  = 4.7° for the haematite component. All three mean directions show a significant increase of the precision parameter k after tilt correction indicating acquisition of the high-temperature magnetization prior to the main folding event in the Jurassic. We explain the difference of the two components of DUN by a process of inclination bias due to compaction to which the platy haematite particles are more susceptible. Applying the elongation-inclination (E/I) method to directional data from over 100 individual samples from location DUN results in a negligible correction for the magnetite component (〈5°), whereas the inclination of the haematite component corrects from –35.0° to –50.3° ( f  = 0.6, error interval –41.4° to –57.9°), which is then equal to the uncorrected magnetite inclination. The small number of samples from section KEL does not allow application of the E/I technique and inclination correction based on high field anisotropy of isothermal remanent magnetization was applied, yielding a corrected inclination of –75.2° ± 4°. Assuming comparable degrees of compaction for both study areas and applying the flattening factor obtained in DUN on samples from KEL, however, would result in comparable inclinations. The identification of inclination shallowing at both sections indicates that the age of magnetization is close to the deposition age. Assuming a reversed polarity of the directions from both areas results in palaeolatitudes of ~30°N for section DUN and ~60°N for the anisotropy-based correction of section KEL. The large difference, however, is geologically very unlikely. The inclination of the magnetite component of DUN (unaffected by inclination shallowing) favours a palaeoposition of ~30°N. This is supported by the inclination shallowing corrected haematite component of DUN yielding a comparable inclination. Therefore, our results indicate that the NTS domain was situated at ~30°N in the early Carboniferous. Furthermore, the NTS zone was probably not connected to Baltica or Siberia prior to the late Palaeozoic.

Description: An exploratory 3-D model of the electrical conductivity structure of the Australian continent is presented. The model is derived from the inversion of vertical magnetic-field transfer functions from the Australia-wide Array of Geomagnetic Stations. Crustal conductivity anomalies evident in the model are consistent with those previously mapped by independent magnetometer array studies and new electrical structures are suggested in the upper mantle. The model represents a seamless continent-scale basis for further models likely to be derived from subsequent studies. The model reveals three upper-mantle enhanced-conductivity anomalies beneath Archaean cratonic regions and two upper-mantle anomalies beneath Phanerozoic terranes in eastern Australia. Two of these anomalies have been investigated by recent magnetotelluric (MT) surveys, one in the Yilgarn Craton–Officer Basin–Musgrave Block the other in the Gawler Craton region, and are consistent with the MT results. Across much of central Australia enhanced conductivity at depths of 50–100 km is observed in the model. This region corresponds well with a recognized seismic velocity gradient at 75–100 km. Conductivity differences are also observed beneath Archaean cratons in Western Australia. The Pilbara Craton is represented as an enhanced conductivity anomaly at about 100 km, corresponding well with the lower-velocity anomaly evident in surface wave tomography models. The Yilgarn Craton is imaged as a low-conductivity body, with conductivity two orders of magnitude lower than the Pilbara Craton, continuing to greater depths.

Description: This paper presents new directional archeomagnetic data from nine Meso-/Neolithic fireplaces, sampled in a cave shelter, at Arconciel, in western Switzerland. Rock magnetic measurements indicate a homogenous magnetic mineralogy in all fireplaces, with magnetite as the main magnetic carrier. The remanent magnetization is stable and generally shows one characteristic directional component. Nine new directions, which were obtained from Arconciel, are combined with 356 other archeomagnetic data from a circular area with a radius of 700 km around this site, to obtain a penalized least square spline fit for the past 9000 yr. We found in general good agreement with other local compilations, such as the Balkan curve, the regional SCHA.DIF.8k model and with lake sediments from UK, Fennoscandia and Switzerland. Nevertheless, a time lag of several centuries is observed for a declination maximum between the archeomagnetic spline fit and the other European data records around 5900 BC. This time lag is also observed in the Swiss lake sediment record; therefore we interpret this shift as a local feature of the Earth's magnetic field.

Description: We present a new approach for 2-D magnetotelluric forward numerical modelling in contrast to traditional numerical methods like finite elements or finite differences. The method used for solving the partial differential equations is based on a mesh-free technique which does not need an underlaying mesh or grid. We use the Meshless Local Petrov–Galerkin (MLPG) method in combination with radial basis functions to simulate the response of a given conductivity model to a plane-wave source. We compare the mesh-free solution with known simulation programs and simple analytical solutions. Furthermore, we discuss the new magnetotelluric modelling method in terms of implementation and stability. First, we study the convergence and discretization errors of the new method with a simple half-space conductivity model. Then we compare our mesh-free simulation results with simple 2-D conductivity models with the results of a well-known finite element program. In the end, we provide a smooth conductivity model calculated with the mesh-free approach. The modelling results, even with randomly distributed nodes, are in a good agreement with those obtained by the finite element method.

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.