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Recent efforts to sequence, survey, and functionally characterize the diverse biosynthetic capabilities of bacteria have identified numerous Biosynthetic Gene Clusters (BGCs). Genes found within BGCs are typically transcriptionally silent, suggesting their expression is tightly regulated. To better elucidate the underlying mechanisms and principles that govern BGC regulation on a DNA sequence level, we employed high-throughput DNA synthesis and multiplexed reporter assays to build and to characterize a library of BGC-derived regulatory sequences. Regulatory sequence transcription levels were measured in the Actinobacteria Streptomyces albidoflavus J1074, a popular model strain from a genus rich in BGC diversity. Transcriptional activities varied over 1000-fold in range and were used to identify key features associated with expression, including GC content, transcription start sites, and sequence motifs. Furthermore, we demonstrated that transcription levels could be modulated through coexpression of global regulatory proteins. Lastly, we developed and optimized a S. albidoflavus cell-free expression system for rapid characterization of regulatory sequences. This work helps to elucidate the regulatory landscape of BGCs and provides a diverse library of characterized regulatory sequences for rational engineering and activation of cryptic BGCs.

Recent advances in cell-free systems have opened up new capabilities in synthetic biology from rapid prototyping of genetic circuits and metabolic pathways to portable diagnostics and biomanufacturing. A current bottleneck in cell-free systems, especially those employing non-E. coli bacterial species, is the required use of plasmid DNA, which can be laborious to construct, clone, and verify. Linear DNA templates offer a faster and more direct route for many cell-free applications, but they are often rapidly degraded in cell-free reactions. In this study, we evaluated GamS from λ-phage, DNA fragments containing Chi-sites, and Ku from Mycobacterium tuberculosis for their ability to protect linear DNA templates in diverse bacterial cell-free systems. We show that these nuclease inhibitors exhibit differential protective activities against endogenous exonucleases in five different cell-free lysates, highlighting their utility for diverse bacterial species. We expect these linear DNA protection strategies will accelerate high-throughput approaches in cell-free synthetic biology.

Microbial communities are a key part to tackling global challenges in human health, environmental conservation, and sustainable agriculture in the coming decade. Recent advances in synthetic biology to study and modify microbial communities have led to important insights into their physiology and ecology. Understanding how targeted changes to microbial communities result in reproducible alterations of the community’s intrinsic fluctuations and function is important for mechanistic reconstruction of microbiomes. Studies of synthetic microbial consortia and comparative analysis of communities in normal and disrupted states have revealed ecological principles that can be leveraged to engineer communities towards desired functions. Tools enabling temporal modulation and sensing of the community dynamics offer precise spatiotemporal control of functions, help to dissect microbial interaction networks, and improve predictions of population temporal dynamics. Here we discuss recent advances to manipulate microbiome dynamics through control of specific strain engraftment and abundance, modulation of cell-cell signaling for tuning population dynamics, infiltration of new functions in the existing community with in situ engineering, and in silico modeling of microbial consortia to predict community function and ecology.

Cell‐free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell‐Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual‐species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell‐free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.

Advances in synthetic biology to build microbes with defined and controllable properties are enabling new approaches to design and program multispecies communities. This emerging field of synthetic ecology will be important for many areas of biotechnology, bioenergy and bioremediation. This endeavor draws upon knowledge from synthetic biology, systems biology, microbial ecology and evolution. Fully realizing the potential of this discipline requires the development of new strategies to control the intercellular interactions, spatiotemporal coordination, robustness, stability and biocontainment of synthetic microbial communities. Here, we review recent experimental, analytical and computational advances to study and build multi-species microbial communities with defined functions and behavior for various applications. We also highlight outstanding challenges and future directions to advance this field.

Microbial communities inhabit our entire planet and have a crucial role in biogeochemical processes, agriculture, biotechnology, and human health. Here, we argue that 'in situ microbiome engineering' represents a new paradigm of community-scale genetic and microbial engineering. We discuss contemporary applications of this approach to directly add, remove, or modify specific sets of functions and alter community-level properties in terrestrial, aquatic, and host-associated microbial communities. Specifically, we highlight emerging in situ genome engineering approaches as tractable techniques to manipulate microbial communities with high specificity and efficacy. Finally, we describe opportunities for technological innovation and ways to bridge existing knowledge gaps to accelerate the development of in situ approaches for microbiome manipulations.

Understanding the complex interactions of protein posttranslational modifications (PTMs) represents a major challenge in metabolic engineering, synthetic biology, and the biomedical sciences. Here, we present a workflow that integrates multiplex automated genome editing (MAGE), genome-scale metabolic modeling, and atomistic molecular dynamics to study the effects of PTMs on metabolic enzymes and microbial fitness. This workflow incorporates complementary approaches across scientific disciplines; provides molecular insight into how PTMs influence cellular fitness during nutrient shifts; and demonstrates how mechanistic details of PTMs can be explored at different biological scales. As a proof of concept, we present a global analysis of PTMs on enzymes in the metabolic network of Escherichia coli. Based on our workflow results, we conduct a more detailed, mechanistic analysis of the PTMs in three proteins: enolase, serine hydroxymethyltransferase, and transaldolase. Application of this workflow identified the roles of specific PTMs in observed experimental phenomena and demonstrated how individual PTMs regulate enzymes, pathways, and, ultimately, cell phenotypes.

Overlapping genes for synthetic biology Overlapping genes yield multiple distinct proteins when translated in alternative reading frames of the same nucleotide sequence. Blazejewski et al. developed a computational algorithm to predict de novo sequence entanglement and experimentally generated functional synthetic overlapping genes. When a sequence of interest was co-encoded with an essential gene in a living bacterium, its evolutionary stability substantially increased. When a gene of interest was synthetically overlapped with a toxin gene, its horizontal gene transfer frequency between bacteria was strongly suppressed. This generalizable strategy for designing, building, and testing overlapping genes helps stabilize vertical gene evolution and restrict horizontal gene flow. Science , this issue p. 595

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is often limited by the use of single-channel reporter assays. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employed this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from >1,000 diverse regulatory sequences revealed functional differences in gene expression that could be predictively modeled. Finally, we constructed and examined the transcriptional capacities of dual-species “hybrid” cell lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic circuit prototyping in new bacterial chassis for synthetic biology.

Robust and predictably performing synthetic circuits rely on the use of well-characterized regulatory parts across different genetic backgrounds and environmental contexts. Here we report the large-scale metagenomic mining of thousands of natural 5' regulatory sequences from diverse bacteria, and their multiplexed gene expression characterization in industrially relevant microbes. We identified sequences with broad and host-specific expression properties that are robust in various growth conditions. We also observed substantial differences between species in terms of their capacity to utilize exogenous regulatory sequences. Finally, we demonstrate programmable species-selective gene expression that produces distinct and diverse output patterns in different microbes. Together, these findings provide a rich resource of characterized natural regulatory sequences and a framework that can be used to engineer synthetic gene circuits with unique and tunable cross-species functionality and properties, and also suggest the prospect of ultimately engineering complex behaviors at the community level.

Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by traditional phosphoramidite column-based approaches. Here, we describe a novel method to amplify oligos from microarray chips for direct use in MAGE to perturb thousands of genomic sites simultaneously. We demonstrated the feasibility of large-scale mutagenesis by inserting T7 promoters upstream of 2585 operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02% per locus with 0.4 average insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.

Comprehensive delineation of complex cellular networks requires high-throughput interrogation of genetic interactions. To address this challenge, we describe the development of a multiplex combinatorial strategy to assess pairwise genetic interactions using CRISPR-Cas9 genome editing and next-generation sequencing. We characterize the performance of combinatorial genome editing and analysis using different promoter and gRNA designs and identified regions of the chimeric RNA that are compatible with next-generation sequencing preparation and quantification. This approach is an important step towards elucidating genetic networks relevant to human diseases and the development of more efficient Cas9-based therapeutics.

Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large‐scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co‐evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo., National Science Foundation (U.S.). Graduate Research Fellowship, Massachusetts Institute of Technology (Neurometrix Presidential Graduate Fellowship)

Recent progress in molecular biology and genetics opens up the possibility of engineering a variety of biological systems, from single-cellular to multicellular organisms. The consortia of microbes that reside on the human body, the human-associated microbiota, are particularly interesting as targets for forward engineering and manipulation due to their relevance in health and disease. New technologies in analysis and perturbation of the human microbiota will lead to better diagnostic and therapeutic strategies against diseases of microbial origin or pathogenesis. Here, we discuss recent advances that are bringing us closer to realizing the true potential of an engineered human-associated microbial community.

Microbial communities that exhibit programmable and modular architecture enable an array of applications in biotechnology and medicine. These engineered synthetic communities can be tuned to sense, degrade, and produce various compounds in both natural and artificial environments. The next phase of synthetic biology will entail engineering of microbes at the level of communities and ecosystems. Here we discuss recent developments in engineering synthetic ecology and future prospects in this emerging area of synthetic biology.

ChIP-seq enables genome-scale identification of regulatory regions that govern gene expression. However, the biological insights generated from ChIP-seq analysis have been limited to predictions of binding sites and cooperative interactions. Furthermore, ChIP-seq data often poorly correlate with in vitro measurements or predicted motifs, highlighting that binding affinity alone is insufficient to explain transcription factor (TF)-binding in vivo. One possibility is that binding sites are not equally accessible across the genome. A more comprehensive biophysical representation of TF-binding is required to improve our ability to understand, predict, and alter gene expression. Here, we show that genome accessibility is a key parameter that impacts TF-binding in bacteria. We developed a thermodynamic model that parameterizes ChIP-seq coverage in terms of genome accessibility and binding affinity. The role of genome accessibility is validated using a large-scale ChIP-seq dataset of the M. tuberculosis regulatory network. We find that accounting for genome accessibility led to a model that explains 63% of the ChIP-seq profile variance, while a model based in motif score alone explains only 35% of the variance. Moreover, our framework enables de novo ChIP-seq peak prediction and is useful for inferring TF-binding peaks in new experimental conditions by reducing the need for additional experiments. We observe that the genome is more accessible in intergenic regions, and that increased accessibility is positively correlated with gene expression and anti-correlated with distance to the origin of replication. Our biophysically motivated model provides a more comprehensive description of TF-binding in vivo from first principles towards a better representation of gene regulation in silico, with promising applications in systems biology., Author Summary A quantitative description of transcription factor (TF) binding in vivo is critical for our understanding of gene regulation. Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) provides a genome-scale map of TF-binding. However, a quantitative characterization of the impact of genome accessibility on TF-binding in bacteria remains elusive. In order to help recruit or block gene expression, TFs must have physical access to regulatory regions. This paper presents a thermodynamics model that describes TF-binding in terms of genome accessibility and binding site affinity. We apply this model in a ChIP-seq dataset for Mycobacterium tuberculosis and observed that genome accessibility is critical to our understanding of TF-binding in vivo. This new model provides practical applications, such as de novo prediction of TF-binding peaks and a framework to measure DNA accessibility from ChIP-seq data. Our model enables us to quantify the relationship of genome accessibility with genomic features and suggest mechanisms that influence genome accessibility in vivo (e.g. distance to oriC). The model proposed in this study gives new perspective for ChIP-seq analysis in bacteria towards an improved description of gene regulation in silico.

Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced in vitro reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein complexes and pathways from the model organism Escherichia coli to reconstitute functional synthetic proteomes in vitro. By application of over 110 MAGE cycles, we successfully inserted hexa-histidine sequences into 38 essential genes in vivo that encode for the entire translation machinery. Streamlined co-purification and reconstitution of the translation protein complex enabled protein synthesis in vitro. Our approach can be applied to a growing area of applications in in vitro one-pot multi-enzyme catalysis (MEC) to manipulate or enhance in vitro pathways such as natural product or carbohydrate biosynthesis.

Engineering at the scale of whole genomes requires fundamentally new molecular biology tools. Recent advances in recombineering using synthetic oligonucleotides enable the rapid generation of mutants at high efficiency and specificity and can be implemented at the genome scale. With these techniques, libraries of mutants can be generated, from which individuals with functionally useful phenotypes can be isolated. Furthermore, populations of cells can be evolved in situ by directed evolution using complex pools of oligonucleotides. Here, we discuss ways to utilize these multiplexed genome engineering methods, with special emphasis on experimental design and implementation.

Engineering at the scale of whole genomes requires fundamentally new molecular biology tools. Recent advances in recombineering using synthetic oligonucleotides enable the rapid generation of mutants at high efficiency and specificity and can be implemented at the genome scale. With these techniques, libraries of mutants can be generated, from which individuals with functionally useful phenotypes can be isolated. Furthermore, populations of cells can be evolved in situ by directed evolution using complex pools of oligonucleotides. Here, we discuss ways to utilize these multiplexed genome engineering methods, with special emphasis on experimental design and implementation.

A genome synthesized entirely from scratch has been used to replace the native genome of a living cell, thus creating a new cell. This achievement marks a new frontier in synthetic biology to design and create genomes for organisms with few genetic tools and for applications in areas of energy, health care and the environment.

This review provides an overview of methodologies and technologies enabling genome-scale engineering, focusing on the design, construction, and testing of modified genomes in a variety of organisms. Future applications for systems and synthetic biology are discussed., Genome-modification technologies enable the rational engineering and perturbation of biological systems. Historically, these methods have been limited to gene insertions or mutations at random or at a few pre-defined locations across the genome. The handful of methods capable of targeted gene editing suffered from low efficiencies, significant labor costs, or both. Recent advances have dramatically expanded our ability to engineer cells in a directed and combinatorial manner. Here, we review current technologies and methodologies for genome-scale engineering, discuss the prospects for extending efficient genome modification to new hosts, and explore the implications of continued advances toward the development of flexibly programmable chasses, novel biochemistries, and safer organismal and ecological engineering.

Microbial ecosystems play an important role in nature. Engineering these systems for industrial, medical, or biotechnological purposes are important pursuits for synthetic biologists and biological engineers moving forward. Here, we provide a review of recent progress in engineering natural and synthetic microbial ecosystems. We highlight important forward engineering design principles, theoretical and quantitative models, new experimental and manipulation tools, and possible applications of microbial ecosystem engineering. We argue that simply engineering individual microbes will lead to fragile homogenous populations that are difficult to sustain, especially in highly heterogeneous and unpredictable environments. Instead, engineered microbial ecosystems are likely to be more robust and able to achieve complex tasks at the spatial and temporal resolution needed for truly programmable biology.