Synthetic Biology: Engineering Living Systems from Biophysical Principles.

Synthetic biology was founded as a biophysical discipline that sought explanations for the origins of life from chemical and physical first principles. Modern synthetic biology has been reinvented as an engineering discipline to design new organisms as well as to better understand fundamental biological mechanisms. However, success is still largely limited to the laboratory and transformative applications of synthetic biology are still in their infancy. Here, we review six principles of living systems and how they compare and contrast with engineered systems. We cite specific examples from the synthetic biology literature that illustrate these principles and speculate on their implications for further study. To fully realize the promise of synthetic biology, we must be aware of life's unique properties.

High phosphate feeding promotes mineral and bone abnormalities in mice with chronic kidney disease.

BACKGROUND: Chronic kidney disease-mineral bone disorder (CKD-MBD) is a systemic syndrome characterized by imbalances in mineral homeostasis, renal osteodystrophy (ROD) and ectopic calcification. The mechanisms underlying this syndrome in individuals with chronic kidney disease (CKD) are not yet clear. METHODS: We examined the effect of normal phosphate (NP) or high phosphate (HP) feeding in the setting of CKD on bone pathology, serum biochemistry and vascular calcification in calcification-prone dilute brown non-agouti (DBA/2) mice. RESULTS: In both NP and HP-fed CKD mice, elevated serum parathyroid hormone and alkaline phosphatase (ALP) levels were observed, but serum phosphorus levels were equivalent compared with sham controls. CKD mice on NP diet showed trabecular alterations in the long bone consistent with high-turnover ROD, including increased trabecular number with abundant osteoblasts and osteoclasts. Despite trabecular bone and serum biochemical changes, CKD/NP mice did not develop vascular calcification. In contrast, CKD/HP mice developed arterial medial calcification (AMC), more severe trabecular bone alterations and cortical bone abnormalities that included decreased cortical thickness and density, and increased cortical porosity. Cortical bone porosity and trabecular number strongly correlated with the degree of aortic calcification. CONCLUSIONS: HP feeding was required to induce the full spectrum of CKD-MBD symptoms in CKD mice.

Computational tools for metabolic engineering.

A great variety of software applications are now employed in the metabolic engineering field. These applications have been created to support a wide range of experimental and analysis techniques. Computational tools are utilized throughout the metabolic engineering workflow to extract and interpret relevant information from large data sets, to present complex models in a more manageable form, and to propose efficient network design strategies. In this review, we present a number of tools that can assist in modifying and understanding cellular metabolic networks. The review covers seven areas of relevance to metabolic engineers. These include metabolic reconstruction efforts, network visualization, nucleic acid and protein engineering, metabolic flux analysis, pathway prospecting, post-structural network analysis and culture optimization. The list of available tools is extensive and we can only highlight a small, representative portion of the tools from each area.

In-Fusion BioBrick assembly and re-engineering.

Genetic circuits can be assembled from standardized biological parts called BioBricks. Examples of BioBricks include promoters, ribosome-binding sites, coding sequences and transcriptional terminators. Standard BioBrick assembly normally involves restriction enzyme digestion and ligation of two BioBricks at a time. The method described here is an alternative assembly strategy that allows for two or more PCR-amplified BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit. This method allows for a large number of parallel assemblies to be performed and is a flexible way to mix and match BioBricks. In-Fusion assembly can be semi-standardized by the use of simple primer design rules that minimize the time involved in planning assembly reactions. We describe the success rate and mutation rate of In-Fusion assembled genetic circuits using various homology and primer lengths. We also demonstrate the success and flexibility of this method with six specific examples of BioBrick assembly and re-engineering. These examples include assembly of two basic parts, part swapping, a deletion, an insertion, and three-way In-Fusion assemblies.

Tyto: A Python Tool Enabling Better Annotation Practices for Synthetic Biology Data-Sharing.

As synthetic biology becomes increasingly automated and data-driven, tools that help researchers implement FAIR (findable-accessible-interoperable-reusable) data management practices are needed. Crucially, in order to support machine processing and reusability of data, it is important that data artifacts are appropriately annotated with metadata drawn from controlled vocabularies. Unfortunately, adopting standardized annotation practices is difficult for many research groups to adopt, given the set of specialized database science skills usually required to interface with ontologies. In response to this need, Take Your Terms from Ontologies (Tyto) is a lightweight Python tool that supports the use of controlled vocabularies in everyday scripting practice. While Tyto has been developed for synthetic biology applications, its utility may extend to users working in other areas of bioinformatics research as well. Tyto is available as a Python package distribution or available as source at https://github.com/SynBioDex/tyto.

Organizing genome engineering for the gigabase scale.

Genome-scale engineering holds great potential to impact science, industry, medicine, and society, and recent improvements in DNA synthesis have enabled the manipulation of megabase genomes. However, coordinating and integrating the workflows and large teams necessary for gigabase genome engineering remains a considerable challenge. We examine this issue and recommend a path forward by: 1) adopting and extending existing representations for designs, assembly plans, samples, data, and workflows; 2) developing new technologies for data curation and quality control; 3) conducting fundamental research on genome-scale modeling and design; and 4) developing new legal and contractual infrastructure to facilitate collaboration.

ShortBOL: A Language for Scripting Designs for Engineered Biological Systems Using Synthetic Biology Open Language (SBOL).

The Synthetic Biology Open Language (SBOL) is an emerging synthetic biology data exchange standard, designed primarily for unambiguous and efficient machine-to-machine communication. However, manual editing of SBOL is generally difficult for nontrivial designs. Here, we describe ShortBOL, a lightweight SBOL scripting language that bridges the gap between manual editing, visual design tools, and direct programming. ShortBOL is a shorthand textual language developed to enable users to create SBOL designs quickly and easily, without requiring strong programming skills or visual design tools.

The Synthetic Biology Open Language (SBOL) Version 3: Simplified Data Exchange for Bioengineering.

The Synthetic Biology Open Language (SBOL) is a community-developed data standard that allows knowledge about biological designs to be captured using a machine-tractable, ontology-backed representation that is built using Semantic Web technologies. While early versions of SBOL focused only on the description of DNA-based components and their sub-components, SBOL can now be used to represent knowledge across multiple scales and throughout the entire synthetic biology workflow, from the specification of a single molecule or DNA fragment through to multicellular systems containing multiple interacting genetic circuits. The third major iteration of the SBOL standard, SBOL3, is an effort to streamline and simplify the underlying data model with a focus on real-world applications, based on experience from the deployment of SBOL in a variety of scientific and industrial settings. Here, we introduce the SBOL3 specification both in comparison to previous versions of SBOL and through practical examples of its use.

Phosphate feeding induces arterial medial calcification in uremic mice: role of serum phosphorus, fibroblast growth factor-23, and osteopontin.

Arterial medial calcification is a major complication in patients with chronic kidney disease and is a strong predictor of cardiovascular and all-cause mortality. We sought to determine the role of dietary phosphorus and the severity of uremia on vascular calcification in calcification-prone DBA/2 mice. Severe and moderate uremia was induced by renal ablation of varying magnitudes. Extensive arterial-medial calcification developed only when the uremic mice were placed on a high-phosphate diet. Arterial calcification in the severely uremic mice fed a high-phosphate diet was significantly associated with hyperphosphatemia. Moderately uremic mice on this diet were not hyperphosphatemic but had a significant rise in their serum levels of fibroblast growth factor 23 (FGF-23) and osteopontin that significantly correlated with arterial medial calcification. Although there was widespread arterial medial calcification, there was no histological evidence of atherosclerosis. At early stages of calcification, the osteochondrogenic markers Runx2 and osteopontin were upregulated, but the smooth muscle cell marker SM22alpha decreased in medial cells, as did the number of smooth muscle cells in extensively calcified regions. These findings suggest that phosphate loading and the severity of uremia play critical roles in controlling arterial medial calcification in mice. Further, FGF-23 and osteopontin may be markers and/or inducers of this process.

pySBOL: A Python Package for Genetic Design Automation and Standardization.

This paper presents pySBOL, a software library for computer-aided design of synthetic biological systems in the Python scripting language. This library provides an easy-to-use, object-oriented, application programming interface (API) with low barrier of entry for synthetic biology application developers. The pySBOL library enables reuse of genetic parts and designs through standardized data exchange with biological parts repositories and software tools that communicate using the Synthetic Biology Open Language (SBOL). In addition, pySBOL supports data management of design-build-test-learn workflows for individual laboratories as well as large, distributed teams of synthetic biologists. PySBOL also lets users add custom data to SBOL files to support the specific data requirements of their research. This extensibility helps users integrate software tool chains and develop workflows for new applications. These features and others make the pySBOL library a valuable tool for supporting engineering practices in synthetic biology. Documentation and installation instructions can be found at pysbol2.readthedocs.io .

DNAplotlib: Programmable Visualization of Genetic Designs and Associated Data.

DNAplotlib ( www.dnaplotlib.org ) is a computational toolkit for the programmable visualization of highly customizable, standards-compliant genetic designs. Functions are provided to aid with both visualization tasks and to extract and overlay associated experimental data. High-quality output is produced in the form of vector-based PDFs, rasterized images, and animated movies. All aspects of the rendering process can be easily customized or extended by the user to cover new forms of genetic part or regulation. DNAplotlib supports improved communication of genetic design information and offers new avenues for static, interactive and dynamic visualizations that map and explore the links between the structure and function of genetic parts, devices and systems; including metabolic pathways and genetic circuits. DNAplotlib is cross-platform software developed using Python.

The Synthetic Biology Open Language (SBOL) provides a community standard for communicating designs in synthetic biology.

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 community-driven, 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.

Designing and engineering evolutionary robust genetic circuits.

BACKGROUND: One problem with engineered genetic circuits in synthetic microbes is their stability over evolutionary time in the absence of selective pressure. Since design of a selective environment for maintaining function of a circuit will be unique to every circuit, general design principles are needed for engineering evolutionary robust circuits that permit the long-term study or applied use of synthetic circuits. RESULTS: We first measured the stability of two BioBrick-assembled genetic circuits propagated in Escherichia coli over multiple generations and the mutations that caused their loss-of-function. The first circuit, T9002, loses function in less than 20 generations and the mutation that repeatedly causes its loss-of-function is a deletion between two homologous transcriptional terminators. To measure the effect between transcriptional terminator homology levels and evolutionary stability, we re-engineered six versions of T9002 with a different transcriptional terminator at the end of the circuit. When there is no homology between terminators, the evolutionary half-life of this circuit is significantly improved over 2-fold and is independent of the expression level. Removing homology between terminators and decreasing expression level 4-fold increases the evolutionary half-life over 17-fold. The second circuit, I7101, loses function in less than 50 generations due to a deletion between repeated operator sequences in the promoter. This circuit was re-engineered with different promoters from a promoter library and using a kanamycin resistance gene (kanR) within the circuit to put a selective pressure on the promoter. The evolutionary stability dynamics and loss-of-function mutations in all these circuits are described. We also found that on average, evolutionary half-life exponentially decreases with increasing expression levels. CONCLUSIONS: A wide variety of loss-of-function mutations are observed in BioBrick-assembled genetic circuits including point mutations, small insertions and deletions, large deletions, and insertion sequence (IS) element insertions that often occur in the scar sequence between parts. Promoter mutations are selected for more than any other biological part. Genetic circuits can be re-engineered to be more evolutionary robust with a few simple design principles: high expression of genetic circuits comes with the cost of low evolutionary stability, avoid repeated sequences, and the use of inducible promoters increases stability. Inclusion of an antibiotic resistance gene within the circuit does not ensure evolutionary stability.