5Heterologous gene expression can be a significant burden to cells, consuming resources and 6 causing decreased growth and stability. We describe here an in vivo monitor that tracks E. 7 coli capacity changes in real-time and can be used to assay the burden synthetic constructs and 8 their parts impose. By measuring capacity, construct designs with reduced burden can be 9 identified and shown to predictably outperform less efficient designs, despite having equivalent 10 expression outputs. 11Robust expression of heterologous genes is necessary for many applications in biotechnology and is 12 central to synthetic biology where predictable fine-tuning of expression is typically desired 1-3 . 13However, for engineered bacteria all heterologous expression represents an unnatural load, 14 consuming cellular resources usually allocated to replication, repair and native gene expression 15 ( Figure 1A). Gene expression burden is a well-known phenomenon characterised by decreased 16 growth rates that can predispose synthetic constructs to evolutionary instability and can 17 unexpectedly alter their behaviour 4-10 . Burden presents a major barrier to predictable and stable 18 engineering of cells, yet it is largely an unquantified phenomenon, inferred in most cases by tracking 19 growth rate decline 5, 6, 11 . Recent research has begun to explore burden, demonstrating how its 20 impact varies between different E. coli strains 6, 11 and showing how expression load can be measured 21 in vitro using cell-free extracts 12 . However, an improved way of quantifying how heterologous gene 22 expression imposes burden in vivo has yet to be described, despite the arrival of new models of 23 bacterial growth that outline the importance of expression resources for the cell [13][14][15] . 24To advance in vivo quantification of burden we developed a fluorescence-based method to measure 25 in real-time the gene expression capacity of bacterial genomes. We built integration vectors to insert 26 a 'capacity monitor', a synthetic constitutive green fluorescent protein (GFP) expression cassette, 27 into defined genomic loci of commonly-used E. coli strains ( Figure S1); reasoning that because this 28 cassette lacks regulation, changes in GFP expression due to global expression changes will reflect 29 changes in resource availability 16 . To demonstrate how the capacity monitor improves on using 30 growth rates to assess burden, we measured GFP expression rates from the genome of DH10B E. coli 31 hosting an operon-expressing plasmid induced at different time-points during exponential growth 32 ( Figure 1B). Capacity (determined as GFP production rate per cell) decreases significantly compared 33 to uninduced cells within 30 minutes of construct induction, and this rapid change contrasts with the 34 smaller, slower decreases in growth observed when culture optical density is measured. The fact 35 that capacity changes precede growth rate changes supports the view that decreased expression 36 resources causes growth rate decline and underlin...

Abstract-This paper employs dissipativity theory for the global analysis of limit cycles in particular dynamical systems of possibly high dimension. Oscillators are regarded as open systems that satisfy a particular dissipation inequality. It is shown that this characterization has implications for the global stability analysis of limit cycle oscillations: i) in isolated oscillators, ii) in interconnections of oscillators, and iii) for the global synchrony analysis in interconnections of identical oscillators.Index Terms-Global limit cycle analysis, global synchronization, Hopf and pitchfork bifurcations, networks of oscillators.

The re-use of previously validated designs is critical to the evolution of synthetic biology from a research discipline to an engineering practice. Here we describe the Synthetic Biology Open Language (SBOL), a proposed data standard for exchanging designs within the synthetic biology community. SBOL represents synthetic biology designs in a communitydriven, formalized format for exchange between software tools, research groups and commercial service providers. The SBOL Developers Group has implemented SBOL as an XML/RDF serialization and provides software libraries and specification documentation to help developers implement SBOL in their own software. We describe early successes, including a demonstration of the utility of SBOL for information exchange between several different software tools and repositories from both academic and industrial partners. As a community-driven standard, SBOL will be updated as synthetic biology evolves to provide specific capabilities for different aspects of the synthetic biology workflow.Synthetic biology treats biological organisms as a new technological medium with a unique set of characteristics, such as the ability to self-repair, evolve and replicate. These characteristics create their own engineering challenges, but offer a rich and largely untapped source of potential applications across a broad range of sectors 1,2 . Applications such as biomolecular computing 3 , metabolic engineering 4 , or reconstruction and exploration of natural cell biology 5,6 commonly require the design of new genetically encoded systems. As engineers, synthetic biologists most often base their designs on previously described 'DNA segments' (see Supplementary Table 1 for definitions of selected terms) to meet their design requirements. Reuse of the DNA sequence for these segments involves their exchange between laboratories and their hierarchical composition to form devices and systems with higher level function.Every engineering field relies on a set of 'standards' 7 that practitioners follow to enable the exchange and reuse of designs for 'systems' , 'devices' and 'components' . Similarly, the representation of synthetic biology designs using computer-readable 'data standards' has the potential to facilitate the forward engineering of novel biological systems from previously characterized devices and components. For example, such standards could enable synthetic biology companies to offer catalogs of devices and components by means of computerreadable data sheets, just as modern semiconductor companies do for electronics. Such standards could also enable a synthetic biologist to develop portions of a design using one software tool, refine the design using another tool, and finally transmit it electronically to a colleague or commercial fabrication company.In order for synthetic biology designs to scale up in complexity, researchers will need to make greater use of specialized design tools and parts repositories. Seamless inter-tool communication would, for example, allow the separation of gene...

Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.

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Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematical model, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the use of endogenous miRNAs as elementary components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

Dynamic control of enzyme expression can be an effective strategy to engineer robust metabolic pathways. It allows a synthetic pathway to self-regulate in response to changes in bioreactor conditions or the metabolic state of the host. The implementation of this regulatory strategy requires gene circuits that couple metabolic signals with the genetic machinery, which is known to be noisy and one of the main sources of cell-to-cell variability. One of the unexplored design aspects of these circuits is the propagation of biochemical noise between enzyme expression and pathway activity. In this article, we quantify the impact of a synthetic feedback circuit on the noise in a metabolic product in order to propose design criteria to reduce cell-to-cell variability. We consider a stochastic model of a catalytic reaction under negative feedback from the product to enzyme expression. On the basis of stochastic simulations and analysis, we show that, depending on the repression strength and promoter strength, transcriptional repression of enzyme expression can amplify or attenuate the noise in the number of product molecules. We obtain analytic estimates for the metabolic noise as a function of the model parameters and show that noise amplification/attenuation is a structural property of the model. We derive an analytic condition on the parameters that lead to attenuation of metabolic noise, suggesting that a higher promoter sensitivity enlarges the parameter design space. In the theoretical case of a switch-like promoter, our analysis reveals that the ability of the circuit to attenuate noise is subject to a trade-off between the repression strength and promoter strength.

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