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...

SUMMARYEngineering artificial gene networks from modular components is one of the major goals of synthetic biology. However, the construction of gene networks with predictable functions remains hampered by a lack of suitable components and the fact that assembled networks often require extensive, iterative retrofitting to work as intended. Here we present an approach that couples libraries of diversified components (synthesized with randomized non-essential sequence) with in silico modeling to guide predictable gene network construction without the need for post-hoc tweaking. We demonstrate our approach in S. cerevisiae by synthesizing regulatory promoter libraries and using them to construct feedforward loop networks with different predicted inputoutput characteristics. We then expand our method to produce a synthetic gene network acting as a predictable timer, modifiable by component choice. We utilize this network to control the timing of yeast sedimentation, illustrating how the plug-and-play nature of our design can be readily applied to biotechnology.

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.

The assembly of DNA from small fragments into large constructs has seen significant recent development, becoming a pivotal technology in the ability to implement the vision of synthetic biology. As the cost of whole gene synthesis is decreasing, whole genome synthesis at the other end of the spectrum has expanded our horizons to the prospect of fully engineered synthetic cells. However, the recently proven ability to synthesise genome-scale DNA is at odds with our ability to rationally engineer biological devices, which lags significantly behind. Most work in synthetic biology takes place on an intermediate scale with the combinatorial construction of networks and metabolic pathways from registries of modular biopart components. Implementation for rapid prototyping of engineered biological circuits requires quick and reliable DNA assembly according to specific architectures. It is apparent that DNA assembly is now a limiting technology in advancing synthetic biology. Current techniques employ standardised restriction enzyme assembly protocols such as BioBricks™, BglBricks and Golden Gate methods. Alternatively, sequence-independent overlap techniques, such as In-Fusion™, SLIC and Gibson isothermal assembly are becoming popular for larger assemblies, and in vivo DNA assembly in yeast and bacillus appears adept for chromosome fabrication. It is important to consider how the use of different technologies may impact the outcome of a construction, since the assembly technique can direct the architecture and diversity of systems that can be made. This review provides a critical examination of recent DNA assembly strategies and considers how this important facilitating aspect of synthetic biology may proceed in the future.

Summary G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.

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Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae. Its high strength, purity, and biocompatibility make it of great interest to materials science; however, precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a strain of Komagataeibacter rhaeticus (K. rhaeticus iGEM) that can produce cellulose at high yields, grow in low-nitrogen conditions, and is highly resistant to toxic chemicals. We achieved external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering, and biotechnology.

PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...