The role of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-­‐15 (ADAMTS-­‐15) in Breast Cancer

Breast cancer is the most common cancer in women and in 2008 accounted for 8% of UK cancer related deaths. A poor prognosis is particularly conferred upon individuals with evidence of metastatic breast cancer. With some studies noting that at least 70% of patients dying with breast cancer have evidence of metastatic disease. In order to develop novel therapeutic strategies a greater understanding of breast cancer tumourigenesis and metastasis is required. Metalloproteinases were implicated as key drivers of metastasis through their ability to degrade the components of the extracellular matrix. This perspective is now superseded with evidence highlighting the involvement of metalloproteinases in an array of biological roles, from maintaining tissue homeostasis to angiogenesis, and importantly these roles can have tumour suppressive effects. Several metalloproteinases from the A Disintegrin and Metalloproteinase with thrombospondin motifs (ADAMTS) family are candidate tumour suppressors, including ADAMTS-15. In the context of breast cancer relatively high levels of ADAMTS-15 expression had previously been associated with increased relapse free survival. However the functional consequences of ADAMTS-15 expression in breast cancer are unknown and are the focus of this thesis. ADAMTS-15 reduced the migration of MDA-MB-231 and MCF-7 cells, in a metalloproteinase-independent manner. This anti-migratory effect likely involves syndecan-4, since modulation of syndecan-4 expression and signalling attenuated this effect. In contrast to its effects on cell migration, only wildtype ADAMTS-15 exhibited an anti-angiogenic effect in in vitro and ex vivo models of angiogenesis. In experimental metastasis assays, both ADAMTS-15 and E362A (metalloproteinase inactive form of ADAMTS-15) reduced metastasis of MDAMB- 231 cells to the liver, though paradoxically, ADAMTS-15 but not E362A enhanced lung colonisation. Taken together these studies demonstrate for the first time that extracellular ADAMTS-15 has multiple tissue context-dependent actions on breast tumour pathophysiology, some of which require its proteolytic activity whereas others do not

Developments in the tools and methodologies of synthetic biology.

Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a body of knowledge from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community

Opportunities for engineering Outer Membrane Vesicles (OMVs) using synthetic biology approaches

Gram-negative bacteria naturally shed lipid vesicles, which contain complex molecular cargoes, from their outer membrane. These outer membrane vesicles (OMVs) have important biological functions relating to microbial stress responses, microbiome regulation, and host-pathogen interactions. OMVs are also attractive vehicles for delivering drugs, vaccines, and other therapeutic agents because of their ability to interact with host cells and their natural immunogenic properties. OMVs are also set to have a positive impact on other biotechnological and medical applications including diagnostics, bioremediation, and metabolic engineering. We envision that the field of synthetic biology offers a compelling opportunity to further expand and accelerate the foundational research and downstream applications of OMVs in a range of applications including the provision of OMV-based healthcare technologies. In our opinion, we discuss how current and potential future synergies between OMV research and synthetic biology approaches might help to further accelerate OMV research and real-world applications for the benefit of animal and human health

Promoting Microbiology Education Through the iGEM Synthetic Biology Competition

Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM and all DNA constructs synthesised by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area

Opportunities to accelerate extracellular vesicle research with cell-free synthetic biology

Extracellular vesicles (EVs) are lipid-membrane nanoparticles that are shed or secreted by many different cell types. The extracellular vesicle (EV) research community has rapidly expanded in recent years and are leading efforts to deepen our understanding of EV biological functions in human physiology and pathology. These insights are also providing a foundation on which future EV-based diagnostics and therapeutics are poised to positively impact human health. However, current limitations in our understanding of EV heterogeneity, cargo loading mechanisms and the nascent development of EV metrology are all areas that have been identified as important scientific challenges. The field of synthetic biology is also contending with the challenge of understanding biological complexity as it seeks to combine multidisciplinary scientific knowledge with engineering principles, to build useful and robust biotechnologies in a responsible manner. Within this context, cell-free systems have emerged as a powerful suite of in vitro biotechnologies that can be employed to interrogate fundamental biological mechanisms, including the study of aspects of EV biogenesis, or to act as a platform technology for medical biosensors and therapeutic biomanufacturing. Cell-free gene expression (CFE) systems also enable in vitro protein production, including membrane proteins, and could conceivably be exploited to rationally engineer, or manufacture, EVs loaded with bespoke molecular cargoes for use in foundational or translational EV research. Our pilot data herein, also demonstrates the feasibility of cell-free EV engineering. In this perspective we discuss the opportunities and challenges for accelerating EV research and healthcare applications with cell-free synthetic biology

A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB)

ADAMTS9, a member of the ADAMTS family, in Xenopus development

Extracellular matrix (ECM) remodeling by metalloproteinases is crucial during development. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin type I motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling. The human family includes 19 members. In this study we identified the 19 members of the ADAMTS family in Xenopus laevis and Xenopus tropicalis. Gene identification and a phylogenetic study revealed strong conservation of the ADAMTS family and contributed to a better annotation of the Xenopus genomes. Expression of the entire ADAMTS family was studied from early stages to tadpole stages of Xenopus, and detailed analysis of ADAMTS9 revealed expression in many structures during organogenesis such as neural crest (NC) derivative tissues, the pronephros and the pancreas. Versican, a matrix component substrate of ADAMTS9 shows a similar expression pattern suggesting a role of ADAMTS9 in the remodeling of the ECM in these structures by degradation of versican

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology