TESTING USED ROLLER BEARINGS FOR QUALITY AND SERVICE LIFE

Synthetic biology is a rapidly expanding field at the interface of the engineering and biological sciences which aims to apply rational design principles in biological contexts. Many natural processes utilise regulatory architectures that parallel those found in control and electrical engineering, which has motivated their implementation as part of synthetic biological constructs. Tools based upon control theoretical concepts can be used to design such systems, as well as to guide their experimental realisation. In this paper we provide examples of biological implementations of negative feedback systems, and discuss progress made toward realisation of other feedback and control architectures. We then outline major challenges posed by the design of such systems, particularly focusing on those which are specific to biological contexts and on which feedback control can have a significant impact. We explore future directions for work in the field, including new approaches for theoretical design of biological control systems, the utilisation of novel components for their implementation, and the potential for application of automation and machine-learning approaches to accelerate synthetic biological research

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering

An adaptive laboratory evolution platform for strain construction and engineering parts

Adaptive Laboratory Evolution (ALE) can be used to obtain useful strains for biotechnology applications. A complete platform containing automation, bioinformatics, and biological selection systems can be used to replace the design and build steps in the rational strain engineering process. In this talk, I will describe the different parts of this platform and current use cases applying the platform to strain construction goals. Examples of advanced biological selection systems which have been developed will be highlighted to show how ALE can be used to obtain production strains for a wider set of products and for focusing selective pressures on targeted enzymatic steps. An additional specific focus will be to highlight what a quality-controlled mutation identification pipeline, a mutation database, and higher order interpretations of mutations using biological network analyses provides for such engineering goals. Finally, I will review what is learned with increasing scale from gathering mutation and strain data from ALE experimentation and what it means for synthetic biology

Automated robotic liquid handling assembly of modular DNA devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation

Repository-based plasmid design

There was an explosion in the amount of commercially available DNA in sequence repositories over the last decade. The number of such plasmids increased from 12,000 to over 300,000 among three of the largest repositories: iGEM, Addgene, and DNASU. A challenge in biodesign remains how to use these and other repository-based sequences effectively, correctly, and seamlessly. This work describes an approach to plasmid design where a plasmid is specified as simply a DNA sequence or list of features. The proposed software then finds the most cost-effective combination of synthetic and PCR-prepared repository fragments to build the plasmid via Gibson assembly®. It finds existing DNA sequences in both user-specified and public DNA databases: iGEM, Addgene, and DNASU. Such a software application is introduced and characterized against all post-2005 iGEM composite parts and all Addgene vectors submitted in 2018 and found to reduce costs by 34% versus a purely synthetic plasmid design approach. The described software will improve current plasmid assembly workflows by shortening design times, improving build quality, and reducing costs.Accepted manuscrip

Directed Placement for mVLSI Devices

Continuous-flow microfluidic devices based on integrated channel networks are becoming increasingly prevalent in research in the biological sciences. At present, these devices are physically laid out by hand by domain experts who understand both the underlying technology and the biological functions that will execute on fabricated devices. The lack of a design science that is specific to microfluidic technology creates a substantial barrier to entry. To address this concern, this article introduces Directed Placement, a physical design algorithm that leverages the natural "directedness" in most modern microfluidic designs: fluid enters at designated inputs, flows through a linear or tree-based network of channels and fluidic components, and exits the device at dedicated outputs. Directed placement creates physical layouts that share many principle similarities to those created by domain experts. Directed placement allows components to be placed closer to their neighbors compared to existing layout algorithms based on planar graph embedding or simulated annealing, leading to an average reduction in laid-out fluid channel length of 91% while improving area utilization by 8% on average. Directed placement is compatible with both passive and active microfluidic devices and is compatible with a variety of mainstream manufacturing technologies

Principles of synthetic biology

In synthetic biology, biological cells and processes are dismantled and reassembled to make novel systems that do useful things. Designs are encoded by deoxyribonucleic acid (DNA); DNA makes biological (bio-)parts; bioparts are combined to make devices; devices are built into biological systems. Computers are used at all stages of the Design-Build-Test-Learn cycle, from mathematical modelling through to the use of robots for the automation of assembly and experimentation. Synthetic biology applies engineering principles of standardisation, modularity, and abstraction, enabling fast prototyping and the ready exchange of designs between synthetic biologists working around the world. Like toy building blocks, compatible modular designs enable bioparts to be combined and optimised easily; biopart specifications are shared in open registries. Synthetic biology is made possible due to major advances in DNA sequencing and synthesis technologies, and through knowledge gleaned in the field of systems biology. Systems biology aims to understand biology across scales, from the molecular and cellular, up to tissues and organisms, and describes cells as complex information-processing systems. By contrast, synthetic biology seeks to design and build its own systems. Applications of synthetic biology are wide-ranging but include impacting healthcare to improve diagnosis and make better treatments for disease; it seeks to improve the environment by finding novel ways to clean up pollution, make industrial processes for chemical synthesis sustainable, and remove the need for damaging farming practices by making better fertilisers. Synthetic biology has the potential to change the way we live and help us to protect the future of our planet

Cell-Free Approaches in Synthetic Biology Utilizing Microfluidics

Synthetic biology is a rapidly growing multidisciplinary branch of science which aims to mimic complex biological systems by creating similar forms. Constructing an artificial system requires optimization at the gene and protein levels to allow the formation of entire biological pathways. Advances in cell-free synthetic biology have helped in discovering new genes, proteins, and pathways bypassing the complexity of the complex pathway interactions in living cells. Furthermore, this method is cost-and time-effective with access to the cellular protein factory without the membrane boundaries. The freedom of design, full automation, and mimicking of in vivo systems reveal advantages of synthetic biology that can improve the molecular understanding of processes, relevant for life science applications. In parallel, in vitro approaches have enhanced our understanding of the living system. This review highlights the recent evolution of cell-free gene design, proteins, and cells integrated with microfluidic platforms as a promising technology, which has allowed for the transformation of the concept of bioprocesses. Although several challenges remain, the manipulation of biological synthetic machinery in microfluidic devices as suitable 'homes' for in vitro protein synthesis has been proposed as a pioneering approach for the development of new platforms, relevant in biomedical and diagnostic contexts towards even the sensing and monitoring of environmental issues