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Synthetic biology approaches life from the perspective of an engineer. Standardized and de novo design of genetic parts to subsequently build reproducible and controllable modules, for example, for circuit design, is a key element. To achieve this, natural systems and elements often serve as a blueprint for researchers. Regulation of protein abundance is controlled at DNA, mRNA, and protein levels. Many tools for the activation or repression of transcription or the destabilization of proteins are available, but easy-to-handle minimal regulatory elements on the mRNA level are preferable when translation needs to be modulated. Regulatory RNAs contribute considerably to regulatory networks in all domains of life. In particular, bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Slowly, sRNAs are attracting the interest of using them for broad applications in synthetic biology. Here, we promote a "plug and play" plasmid toolset to quickly and efficiently create synthetic sRNAs to study sRNA biology or their application in bacteria. We propose a simple benchmarking assay by targeting the

DNA replication prior to cell division is essential for the proliferation of all cells. Bacterial chromosomes are replicated bidirectionally from a single origin of replication, with replication proceeding at about 1000 bp per second. For the best-studied model organism, Escherichia coli, this translates into a replication time of about 40 min for its 4.6 Mb chromosome. Nevertheless, E. coli can propagate by overlapping replication cycles with a maximum short doubling time of 20 min. The fastest growing bacterium known today, Vibrio natriegens, is able to replicate with a generation time of less than 10 min. It has a bipartite genome with chromosome sizes of 3.2 and 1.9 Mb. Is simultaneous replication from two origins a prerequisite for its rapid growth? We fused the two chromosomes of V. natriegens to create a strain carrying a 5.2 Mb chromosome with a single origin of replication. Compared to the wild-type, this strain showed little deviation in growth rate. This suggests that the split genome is not a prerequisite for rapid growth, and that DNA replication is not an important growth rate-limiting factor.

SummaryGenomic rearrangements contribute to gene copy number alterations, disruption of protein-coding sequences and/or perturbation of cis-regulatory networks. SCRaMbLE, a Cre/loxP-based system implanted in synthetic yeast chromosomes, can effectively introduce genomic rearrangements, and is thus a potential tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we constructed a yeast strain, SparLox83, containing 83 loxPsym sites distributed across all 16 chromosomes, with at least two sites per chromosome. Inducing Cre recombinase expression in SparLox83 produced versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, SCRaMbLE of the hetero-diploid strains derived from crossing SparLox83 with strains possessing synthetic chromosome III (synIII) from the Sc2.0 project led to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to a strain with only synIII. Analysis of these evolved strains demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and can consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool to study the consequence of genomic rearrangements and help accelerate strain engineering in Saccharomyces cerevisiae.

SUMMARY: Synthetic small RNAs are gaining increasing attention in the field of synthetic biology and bioengineering for efficient post-transcriptional regulation of gene expression. However, the optimal design of synthetic small RNAs is challenging because alterations may impair functions or off-target effects can arise. Here, we introduce DIGGER-Bac, a toolbox for Design and Identification of seed regions for Golden Gate assembly and Expression of synthetic small RNAs in Bacteria. The SEEDling tool predicts optimal small RNA seed regions in combination with user-defined small RNA scaffolds for efficient regulation of specified mRNA targets. Results are passed on to the G-GArden tool, which assists with primer design for high-fidelity Golden Gate assembly of the desired synthetic small RNA constructs. AVAILABILITY AND IMPLEMENTATION: Both SEEDling and G-GArden are freely available at https://github.com/DIGGER-Bac under the CC BY-NC-SA 4.0 license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Nature ecology & evolution 7(5), 756 - 767 (2023). doi:10.1038/s41559-023-02018-8, Highly specific interactions between proteins are a fundamental prerequisite for life, but how they evolve remains an unsolved problem. In particular, interactions between initially unrelated proteins require that they evolve matching surfaces. It is unclear whether such surface compatibilities can only be built by selection in small incremental steps, or whether they can also emerge fortuitously. Here, we used molecular phylogenetics, ancestral sequence reconstruction and biophysical characterization of resurrected proteins to retrace the evolution of an allosteric interaction between two proteins that act in the cyanobacterial photoprotection system. We show that this interaction between the orange carotenoid protein (OCP) and its unrelated regulator, the fluorescence recovery protein (FRP), evolved when a precursor of FRP was horizontally acquired by cyanobacteria. FRP’s precursors could already interact with and regulate OCP even before these proteins first encountered each other in an ancestral cyanobacterium. The OCP–FRP interaction exploits an ancient dimer interface in OCP, which also predates the recruitment of FRP into the photoprotection system. Together, our work shows how evolution can fashion complex regulatory systems easily out of pre-existing components., Published by Nature Publishing Group, London

Optimization of the metabolic flux through heterologous pathways to improve bioproduction or utilization of alternative substrates requires both fine-tuning of non-native gene expression levels and improvement of the host genome. The SCRaMbLE system incorporated into synthetic Sc2.0 yeast strains enables a rapid approach to rearrange the genome ofSaccharomyces cerevisiaein order to create optimized chassis. Here, we show that the light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or circular synthetic chromosome III. We present an efficient and straightforward workflow for the identification of complex rearranged synthetic chromosomes from SCRaMbLEd isolates without selection pressure. The screening method is based on novel genotyping primers, theloxPsymtags, which indicate not only deletions but also inversions and translocations. Long-read Nanopore sequencing is used to decode the selected genotypes and shows in conjunction with flow cytometry that large-scale karyotype alterations can be a consequence of SCRaMbLE.

SUMMARYThe genome of an organism is inherited from its ancestor and keeps evolving over time, however, how much the current version could be altered remains unknown. Here, we use the left arm of chromosome XII (chrXIIL) as an example to probe the genome plasticity in Saccharomyces cerevisiae. A neochromosome was designed to harbor originally dispersed genes. The essentiality of sequences in chrXIIL was dissected by targeted DNA removal, chromosome truncation and random deletion. Notably, 12 genes were sufficient for survival, while 25 genes are required to retain robust fitness. Next, we demonstrated these genes could be reconstructed using synthetic regulatory sequences and recoded open-reading frames with “one-amino-acid-one-codon” strategy. Finally, we built a neochromsome, which could substitute for chrXIIL for cell viability, with these reconstructed genes. Our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional chromosomes with completely artificial sequences.HIGHLIGHTSA neochromosome was designed to facilitate the assembly of exogenous DNA for stable expression in yeastThe left arm of chrXII could be minimized to just 12 genes to maintain viability, but additional genes were required to retain robust fitnessComprehensive recoding and transcriptional refactoring using artificial regulatory sequences produced a functional chromosome armA completely reconstructed neochromosome could replace the chrXIIL to maintain comparable fitness

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs.HighlightsDe novo design, construction and functional characterization of a neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae.Increasing the copy number of the 275 highly expressed tRNA genes causes cellular burden, which the host cell likely buffers either by selecting for partial tRNA neochromosome deletions or by increasing its ploidy.The tRNA neochromosome can be chemically extracted and transformed into new strain backgrounds, enabling its transplantation into multi-synthetic chromosome strains to finalize the Sc2.0 strain.Comprehensive functional characterization does not pinpoint a singular cause for the cellular burden caused by the tRNA neochromosome, but does reveal novel insights into its tRNA and structural chromosome biology.

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE system to the newly synthesized Sc2.0 megabase chromosome VII (synVII), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by i) removing most of the chromosome content, or ii) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic yeast resource opens new paradigms in studying aneuploidy.In briefUse of SCRaMbLE and newly synthesized Mb-scale Sc2.0 chromosome VII enables insights into genotype/phenotype relationships associated with aneuploidyHighlightsDe novo design and synthesis of a Mb-scale synthetic yeast chromosome VII, carrying 11.8% sequence modifications and representing nearly 10% of the yeast genome.A disomic yeast (n + synVII) is constructed for dissecting the aneuploidy phenotypeSCRaMbLE enables systematic exploration of regions causing aneuploidy phenotypesChromosomal copy number and content both contribute to aneuploidy phenotypesA 20 Kb deletion on the right arm of synVII leads to fitness improvement linked to up-regulation of protein synthesis

Recent publications have investigated and demonstrated new digital modulation techniques that allow realization of orthogonal frequency division multiplexing (OFDM) radar with relatively low hardware effort. This is especially interesting as the digitally defined signals offer additional freedom for multiplexing schemes of multiple-input multiple-output (MIMO) radar sensors. MIMO radar sensors rely on strictly orthogonal signals to distinguish between multiple transmitters (TX). To span a virtual array with a maximum number of antenna positions, a novel method that combines orthogonal signals with strongly correlated signals generated by space-time coding (STC) is introduced. This leads to virtual transmit antenna positions that are used to improve the unambiguous angular area of an otherwise ambiguous MIMO antenna array. The proposed method is validated with an integrated OFDM radar at millimeter-wave frequencies.

Amoeboid cell motility is essential for a wide range of biological processes including wound healing, embryonic morphogenesis, and cancer metastasis. It relies on complex dynamical patterns of cell shape changes that pose long-standing challenges to mathematical modeling and raise a need for automated and reproducible approaches to extract quantitative morphological features from image sequences. Here, we introduce a theoretical framework and a computational method for obtaining smooth representations of the spatiotemporal contour dynamics from stacks of segmented microscopy images. Based on a Gaussian process regression we propose a one-parameter family of regularized contour flows that allows us to continuously track reference points (virtual markers) between successive cell contours. We use this approach to define a coordinate system on the moving cell boundary and to represent different local geometric quantities in this frame of reference. In particular, we introduce the local marker dispersion as a measure to identify localized membrane expansions and provide a fully automated way to extract the properties of such expansions, including their area and growth time. The methods are available as an open-source software package called AmoePy, a Python-based toolbox for analyzing amoeboid cell motility (based on time-lapse microscopy data), including a graphical user interface and detailed documentation. Due to the mathematical rigor of our framework, we envision it to be of use for the development of novel cell motility models. We mainly use experimental data of the social amoeba Dictyostelium discoideum to illustrate and validate our approach., Comment: 31 pages, 12 figures, updated version after publication (see 10.1371/journal.pcbi.1009268), for supporting information, see 10.5281/zenodo.3984179, for software and data publication, see 10.5281/zenodo.3982371 and 10.5061/dryad.b5mkkwhbd

Digital modulations such as orthogonal frequency-division multiplexing (OFDM) have received increasing interest for radar. However, the required fast analog-to-digital (AD) and digital-to-analog (DA) converters providing the necessary bandwidth have not been feasible for automotive applications so far. In this paper, the first highly integrated, fully coherent OFDM radar capable of multiple-input multiple-output (MIMO) and stepped-carrier OFDM for automotive applications is presented. The stepped-carrier OFDM approach is adopted to reduce AD/DA requirements using an analog local oscillator (LO) frequency step generator. The capability to generate frequency steps with a duration of a few microseconds required for automotive applications is demonstrated. Calibration steps that are required with the new architecture are discussed and measurements are done to validate the functionality of the system. Also, possibilities for adaptive reconfiguration of the radar sensor in the presence of ambiguities are demonstrated.

BackgroundRedundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction.ResultsWe develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified.ConclusionsWe develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.

We broadened the usage of DNA transposon technology by demonstrating its capacity for the rapid creation of expression libraries for long biochemical pathways, which is beyond the classical application of building genome-scale knockout libraries in yeasts. This strategy efficiently leverages the readily available fine-tuning impact provided by the diverse transcriptional environment surrounding each random integration locus. We benchmark the transposon-mediated integration against the nonhomologous end joining-mediated strategy. The latter strategy was demonstrated for achieving pathway random integration in other yeasts but is associated with a high false-positive rate in the absence of a high-throughput screening method. Our key innovation of a nonreplicable circular DNA platform increased the possibility of identifying top-producing variants to 97%. Compared to the classical DNA transposition protocol, the design of a nonreplicable circular DNA skipped the step of counter-selection for plasmid removal and thus not only reduced the time required for the step of library creation from 10 to 5 d but also efficiently removed the "transposition escapers", which undesirably represented almost 80% of the entire population as false positives. Using one endogenous product (i.e., shikimate) and one heterologous product (i.e., (S)-norcoclaurine) as examples, we presented a streamlined procedure to rapidly identify high-producing variants with titers significantly higher than the reported data in the literature. We selected Scheffersomyces stipitis, a representative nonconventional yeast, as a demo, but the strategy can be generalized to other nonconventional yeasts. This new exploration of transposon technology, therefore, adds a highly versatile tool to accelerate the development of novel species as microbial cell factories for producing value-added chemicals.

Orthogonal frequency-division multiplexing is a promising modulation format for future automotive radar sensors. Although it benefits from a simple homodyne transceiver design, it requires quadrature signal generation and evaluation, which suffers from IQ-imbalance at transmitter and receiver. This paper describes the effects of IQ-imbalance on OFDM radars in general and demonstrates how digital signal processing techniques can be adopted to mitigate this impairment based on adaptive filtering. The effectiveness of the approach is demonstrated with simulations.

Excavations at Huqoq in Israel’s eastern Lower Galilee are bringing to light a Late Roman synagogue, a medieval public building, and the remains of ancient and modern (pre-1948) villages. In this interim report, we describe the major discoveries of the 2014–2017 seasons, including the extraordinary figural mosaics decorating the synagogue floor. Our discoveries provide evidence of a Galilean Jewish community that flourished through the 5th and 6th centuries c.e.—a picture contrasting with recent claims of a decline in Jewish settlement under Byzantine Christian rule. The possibility that the medieval public building might also be a synagogue has important implications for understanding Galilean Jewish settlement in the Middle Ages, about which almost nothing is known. The excavations also shed light on the last phase of the settlement’s long history: the development of the modern village of Yakuk in the 19th through 20th centuries., peer-reviewed

The requirement of fast analog-to-digital (AD) and digital-to-analog (DA) converters is a challenge for high-resolution orthogonal frequency division multiplexing (OFDM) radars. This paper introduces a method to increase the modulation bandwidth of an OFDM signal by using an additional frequency modulated continuous wave carrier to reduce the requirements for the AD/DA converters while maintaining a high unambiguous velocity range. Multiplexing of transmit antennas with OFDM is still possible with the combined modulation. The presented approach is verified by simulations and measurements. A reduction of AD/DA converter bandwidth by factor eight is achieved.

Recent publications show that the potential of using orthogonal frequency division multiplexing waveforms as radar signals. Since the range resolution is proportional to the RF bandwidth, the major obstacle that obstructs the practical use in automotive and other low-cost radars is the requirement to sample the received signal at sampling rates that span the whole RF signal bandwidth requiring ADCs with sampling rates in the order of GHz. This paper presents a method to achieve the high range resolution induced by a large RF bandwidth, but with a much lower baseband bandwidth, consequently requiring a much slower ADC while at the same time delivering a velocity profile for each subcarrier. In addition, the processing scheme induces a range migration compensation, independent of the number of targets. This is achieved with barely increased computational effort. The scheme is verified with simulations and measurements at 77 GHz.

Recent publications show that major limitations of orthogonal frequency-division multiplexing (OFDM) as modulation format for automotive radars are solved. These include the requirements on analog-to-digital and digital-to-analog converters whose required sampling rate can be reduced using a stepped-carrier OFDM-scheme as well as the all-cell-Doppler-correction-based approach that counteracts the loss of orthogonality due to the Doppler shift. However, so far, the application of both methods is exclusive to each other. In this paper, a combined evaluation scheme is presented that allows to use a stepped-carrier scheme in combination with a quasi repeated-symbol OFDM-waveform and includes the Doppler-correction into the DFT for velocity estimation. This yields a high resolution radar image in both range and velocity at low sampling rate with significantly increased signal-to-noise ratio (SNR) as the orthogonality of subcarriers is preserved. The scheme is verified with simulations that demonstrate its benefits.

In many applications, the direction of arrival (DoA) information of the radar signal plays a decisive role in target localization. A multiple-input multiple-output (MIMO) radar allows to obtain the position of an object in space within one measurement frame. Recent research and publications verify the high potential of digital radar principles such as orthogonal frequency-division multiplexing (OFDM). In this letter, a MIMO-OFDM approach based on random frequency and time-division multiplexing is presented. It is enhanced by a multidimensional compressed sensing method which utilizes the information of multiple channels. The approach is validated and compared to other MIMO-OFDM approaches using measurements of an experimental radar at 72.5 GHz, acceptedVersion

The deliverable D4.7 concerns the work achieved by IRCAM until M36 for the “auto-tagging of music”. The deliverable is a research report. The software libraries resulting from the research have been integrated into Fincons/HearDis! Music Library Manager or are used by TU Berlin. The final software libraries are described in D4.5. The research work on auto-tagging has concentrated on four aspects: 1) Further improving IRCAM’s machine-learning system ircamclass. This has been done by developing the new MASSS audio features, including audio augmentation and audio segmentation into ircamclass. The system has then been applied to train HearDis! “soft” features (Vocals-1, Vocals-2, Pop-Appeal, Intensity, Instrumentation, Timbre, Genre, Style). This is described in Part 3. 2) Developing two sets of “hard” features (i.e. related to musical or musicological concepts) as specified by HearDis! (for integration into Fincons/HearDis! Music Library Manager) and TU Berlin (as input for the prediction model of the GMBI attributes). Such features are either derived from previously estimated higher-level concepts (such as structure, key or succession of chords) or by developing new signal processing algorithm (such as HPSS) or main melody estimation. This is described in Part 4. 3) Developing audio features to characterize the audio quality of a music track. The goal is to describe the quality of the audio independently of its apparent encoding. This is then used to estimate audio degradation or music decade. This is to be used to ensure that playlists contain tracks with similar audio quality. This is described in Part 5. 4) Developing innovative algorithms to extract specific audio features to improve music mixes. So far, innovative techniques (based on various Blind Audio Source Separation algorithms and Convolutional Neural Network) have been developed for singing voice separation, singing voice segmentation, music structure boundaries estimation, and DJ cue-region estimation. This is described in Part 6.

Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed “essential raft”, where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast.

Highlights • Viral, organelle and bacterial genomes can be synthesized. • Sc2.0 is on the way to generating the first synthetic designer eukaryote. • 6 of 17 S. cerevisiae chromosomes are synthesized and published. • GP-write will open a new era of synthetic genomics., Since the first synthetic gene was synthesized in 1970s, the efficiency and the capacity of made-to-order DNA sequence synthesis has increased by several orders of magnitude. Advances in DNA synthesis and assembly over the past years has resulted in a steep drop in price for custom made DNA. Similar effects were observed in DNA sequencing technologies which underpin DNA-reading projects. Today, synthetic DNA sequences with more than 10 000 bps and turn-around times of a few weeks are commercially available. This enables researchers to perform large-scale projects to write synthetic chromosomes and characterize their functionalities in vivo. Synthetic genomics opens up new paradigms to study the genome fundamentals and engineer novel biological functions.

The advances in digital modulations for radar, like OFDM, show promising results. These modulation concepts benefit from high integration of analog and digital CMOS technology, as they rely on digital signal generation and processing. In this paper, a two-channel OFDM single sideband transmitter in CMOS SOI technology is presented. The chip performance is evaluated and broadband sideband suppression of the OFDM symbols using digital predistortion is demonstrated.

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. Questions of how chromosomes need to be constructed to maintain the genetic information can now be answered by a learning-by-building approach. Here, we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular Modular Cloning system (MoClo).

Orthogonal frequency-division multiplexing (OFDM) is a promising candidate for future digital-centric radar systems. One major limitation for practical implementations is the required sampling rate in the order of GHz, which can be solved by using a stepped-carrier technique in combination with OFDM signals of smaller bandwidth, theoretically resulting in the same resolution as a conventional OFDM scheme. However, a signal source capable of generating such a carrier has to fulfill several constraints. In this paper, the effects of deterministic and random inaccuracies of the carrier generation as well as effects of phase noise are described and evaluated. A transient model that describes the switching between carriers is given and all effects are verified using a radar demonstrator at 77 GHz.

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. Questions of how chromosomes need to be constructed to maintain the genetic information can now be answered by a learning-by-building approach. Here, we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular Modular Cloning system (MoClo).

In this article, we consider a recent proposal that Jewish settlement in eastern Galilee experienced a demographic crisis in the late third century and first half of the fourth century c.e. This proposal is based largely on pottery that was collected during intensive surveys of sites in eastern lower Galilee, supplemented by limited data from excavations. However, the ambiguous nature of the survey data means that this material can be interpreted to support a different settlement picture. The crux of our disagreement hinges on the dating and interpretation of the ceramic evidence and the use of survey data as a basis for sweeping historical reconstructions. To demonstrate these points, we discuss three sites that were surveyed (Nasr ed-Din, Parod, and Horvat Ammudim) and analyze selected deposits from the excavations at Capernaum and Meiron. We conclude that the data from these sites can be interpreted as supporting a picture of continuous settlement through the fourth, fifth, and sixth centuries, even al...

What a living organism looks like and how it works and what are its components-all this is encoded on DNA, the genetic blueprint. Consequently, the way to change an organism is to change its genetic information. Since the first pieces of recombinant DNA have been used to transform cells in the 1970s, this approach has been enormously extended. Bigger and bigger parts of the genetic information have been exchanged or added over the years. Now we are at a point where the construction of entire chromosomes becomes a reachable goal and first examples appear. This development leads to fundamental new questions, for example, about what is possible and desirable to build or what construction rules one needs to follow when building synthetic chromosomes. Here we review the recent progress in the field, discuss current challenges and speculate on the appearance of future synthetic chromosomes.

This article enhances our understanding of how the oil lamps found throughout the ancient Near East were used by the ancients. By testing two kinds of replica oil lamps (open and closed), the autho...

Regulators of DNA replication in bacteria are an attractive target for new antibiotics, as not only is replication essential for cell viability, but its underlying mechanisms also differ from those operating in eukaryotes. The genetic information of most bacteria is encoded on a single chromosome, but about 10% of species carry a split genome spanning multiple chromosomes. The best studied bacterium in this context is the human pathogen Vibrio cholerae, with a primary chromosome (Chr1) of 3 M bps, and a secondary one (Chr2) of about 1 M bps. Replication of Chr2 is under control of a unique mechanism, presenting a potential target in the development of V. cholerae-specific antibiotics. A common challenge in such endeavors is whether the effects of candidate chemicals can be focused on specific mechanisms, such as DNA replication. To test the specificity of antimicrobial substances independent of other features of the V. cholerae cell for the replication mechanism of the V. cholerae secondary chromosome, we establish the replication machinery in the heterologous E. coli system. We characterize an E. coli strain in which chromosomal replication is driven by the replication origin of V. cholerae Chr2. Surprisingly, the E. coli ori2 strain was not inhibited by vibrepin, previously found to inhibit ori2-based replication.

The evaluation of radar scenes with high dynamic range is a challenge for radar systems. In frequency modulated continuous wave (FMCW) based systems, dynamic range can be improved by applying a high-pass filter after down conversion. If a different modulation scheme (such as OFDM) is used, the maximum achieveable dynamic range of the system is limited. In this paper antenna polarization modulation (APM) is introduced to eliminate this weakness. It can be applied in addition to an arbitrary radar modulation while no extra Tx or Rx paths are required. The system can be used to either improve dynamic range, evaluate polarimetric targets or refine the detectable radar scene.

Filamentous ascomycetes have long been known as producers of a variety of secondary metabolites, many of which have toxic effects on other organisms. However, the role of these metabolites in the biology of the fungi that produce them remains in most cases enigmatic. A major group of fungal secondary metabolites are polyketides. They are chemically diverse, but have in common that their chemical scaffolds are synthesized by polyketide synthases (PKSs). In a previous study, we analyzed development-dependent expression of pks genes in the filamentous ascomycete Sordaria macrospora. Here, we show that a deletion mutant of the pks4 gene is sterile, producing only protoperithecia but no mature perithecia, whereas overexpression of pks4 leads to enlarged, malformed fruiting bodies. Thus, correct expression levels of pks4 are essential for wild type-like perithecia formation. The predicted PKS4 protein has a domain structure that is similar to homologs in other fungi, but conserved residues of a methyl transferase domain present in other fungi are mutated in PKS4. Expression of several developmental genes is misregulated in the pks4 mutant. Surprisingly, the development-associated app gene is not downregulated in the mutant, in contrast to all other previously studied mutants with a block at the protoperithecial stage. Our data show that the polyketide synthase gene pks4 is essential for sexual development and plays a role in regulating fruiting body morphology.

Learning by building is one of the core ideas of synthetic biology research. Consequently, building synthetic chromosomes is the way to fully understand chromosome characteristics. The last years have seen exciting synthetic chromosome studies. We had previously introduced the synthetic secondary chromosome synVicII in Escherichia coli. It is based on the replication mechanism of the secondary chromosome in Vibrio cholerae. Here, we present a detailed analysis of its genetic characteristics and a selection approach to optimize replicon stability. We probe the origin diversity of secondary chromosomes from Vibrionaceae by construction of several new respective replicons. Finally, we present a synVicII version 2.0 with several innovations including its full compatibility with the popular modular cloning (MoClo) assembly system.