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© 2019 The Authors. ; While prokaryotic promoters controlled by signal‐responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription—as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation‐inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo. ; This work was funded by the SETH Project of the Spanish Ministry of Science RTI2018‐095584‐B‐C42, MADONNA (H2020‐FET‐OPEN‐RIA‐2017‐1‐766975), BioRoboost (H2020‐NMBP‐BIO‐CSA‐2018) and SYNBIO4FLAV (H2020‐NMBP/0500) Contracts of the European Union, the S2017/BMD‐3691 InGEMICS‐CM funded by the Comunidad de Madrid (European Structural and Investment Funds) and the SynBio3D project of the UK Engineering and Physical Sciences Research Council (EP/R019002/1).

This article belongs to the Special Issue Synthetic Biology: From Living Computers to Terraformation. ; We present a scheme for implementing a version of task switching in engineered bacteria, based on the manipulation of plasmid copy numbers. Our method allows for the embedding of multiple computations in a cellular population, whilst minimising resource usage inefficiency. We describe the results of computational simulations of our model, and discuss the potential for future work in this area. ; The work of A.G.-M. is supported by the SynBio3D (UK-EPSRC-EP/R019002/1) project of the UK Engineering and Physical Sciences Research Council and the BioRoboost (EU-H2020-BIOTEC-820699) Contract of the European Union. The work of AR-P is supported by the Spanish TIN2016-81079-R, (MINECO AEI/FEDER, EU) and Madrid Gov. project B2017/BMD-3691, InGEMICS-CM (FSE/FEDER, EU). Work in FdlC laboratory was financed by grant BFU2017-86378-P from the Ministry of Science and Technology (Spain). ; Peer reviewed

As synthetic biology moves away from trial and error and embraces more formal processes, workflows have emerged that cover the roadmap from conceptualization of a genetic device to its construction and measurement. This latter aspect (i.e., characterization and measurement of synthetic genetic constructs) has received relatively little attention to date, but it is crucial for their outcome. An end-to-end use case for engineering a simple synthetic device is presented, which is supported by information standards and computational methods and focuses on such characterization/measurement. This workflow captures the main stages of genetic device design and description and offers standardized tools for both population-based measurement and single-cell analysis. To this end, three separate aspects are addressed. First, the specific vector features are discussed. Although device/circuit design has been successfully automated, important structural information is usually overlooked, as in the case of plasmid vectors. The use of the Standard European Vector Architecture (SEVA) is advocated for selecting the optimal carrier of a design and its thorough description in order to unequivocally correlate digital definitions and molecular devices. A digital version of this plasmid format was developed with the Synthetic Biology Open Language (SBOL) along with a software tool that allows users to embed genetic parts in vector cargoes. This enables annotation of a mathematical model of the device's kinetic reactions formatted with the Systems Biology Markup Language (SBML). From that point onward, the experimental results and their in silico counterparts proceed alongside, with constant feedback to preserve consistency between them. A second aspect involves a framework for the calibration of fluorescence-based measurements. One of the most challenging endeavors in standardization, metrology, is tackled by reinterpreting the experimental output in light of simulation results, allowing us to turn arbitrary fluorescence units into relative measurements. Finally, integration of single-cell methods into a framework for multicellular simulation and measurement is addressed, allowing standardized inspection of the interplay between the carrier chassis and the culture conditions. ; This work was supported by the CAMBIOS Project of the Spanish Ministry of Economy and Competitiveness; the EVOPROG, ARISYS, and EMPOWERPUTIDA Contracts from the European Union; and the PROMT Project of the Madrid Regional Government to V.d.L. ; Peer reviewed

Synthetic biology uses living cells as the substrate for performing human-defined computations. Many current implementations of cellular computing are based on the "genetic circuit" metaphor, an approximation of the operation of silicon-based computers. Although this conceptual mapping has been relatively successful, we argue that it fundamentally limits the types of computation that may be engineered inside the cell, and fails to exploit the rich and diverse functionality available in natural living systems. We propose the notion of "cellular supremacy" to focus attention on domains in which biocomputing might offer superior performance over traditional computers. We consider potential pathways toward cellular supremacy, and suggest application areas in which it may be found. ; A.G.-M. was supported by the SynBio3D project of the UK Engineering and Physical Sciences Research Council (EP/R019002/1) and the European CSA on biological standardization BIOROBOOST (EU grant number 820699). T.E.G. was supported by a Royal Society University Research Fellowship (grant UF160357) and BrisSynBio, a BBSRC/ EPSRC Synthetic Biology Research Centre (grant BB/L01386X/1). P.Z. was supported by the EPSRC Portabolomics project (grant EP/N031962/1). P.C. was supported by SynBioChem, a BBSRC/EPSRC Centre for Synthetic Biology of Fine and Specialty Chemicals (grant BB/M017702/1) and the ShikiFactory100 project of the European Union's Horizon 2020 research and innovation programme under grant agreement 814408.

© The Author(s) 2019. ; The Standard European Vector Architecture 3.0 database (SEVA-DB 3.0, http://seva.cnb.csic.es) is the update of the platform launched in 2013 both as a web-based resource and as a material repository of formatted genetic tools (mostly plasmids) for analysis, construction and deployment of complex bacterial phenotypes. The period between the first version of SEVA-DB and the present time has witnessed several technical, computational and conceptual advances in genetic/genomic engineering of prokaryotes that have enabled upgrading of the utilities of the updated database. Novelties include not only a more user-friendly web interface and many more plasmid vectors, but also new links of the plasmids to advanced bioinformatic tools. These provide an intuitive visualization of the constructs at stake and a range of virtual manipulations of DNA segments that were not possible before. Finally, the list of canonical SEVA plasmids is available in machine-readable SBOL (Synthetic Biology Open Language) format. This ensures interoperability with other platforms and affords simulations of their behaviour under different in vivo conditions. We argue that the SEVA-DB will remain a useful resource for extending Synthetic Biology approaches towards non-standard bacterial species as well as genetically programming new prokaryotic chassis for a suite of fundamental and biotechnological endeavours. ; The SEVA repository has been developed with funds of the SETH Project of the Spanish Ministry of Science [RTI 2018-095584-B-C42, MADONNA (H2020-FETOPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBPBIO-CSA-2018), SYNBIO4FLAV (H2020-NMBP/0500) and LIAR (H2020-EU.1.2.1-686585)]; Contracts of the European Union and the S2017/BMD-3691 InGEMICS-CM funded by the Comunidad de Madrid (European Structural and Investment Funds) and the UK Engineering and Physical Science Research Council [EP/R019002/1]. Work at Raytheon was supported by the Air Force Research Laboratory (AFRL) and DARPA under contract FA875017CO184. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the AFRL and DARPA.This document does not contain technology or technical data controlled under either U.S. International Traffic in Arms Regulation or U.S. Export Administration Regulations. Funding for open access charge: H2020-NMBP-BIO-CSA-2018.