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 This perspective is now superseded with evidence highlighting the involvement of metalloproteinases in an array of biological roles, from maintaining tissue homeostasis to angiogenesis, and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

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

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

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

The roles of ADAMTS metalloproteinases in tumorigenesis and metastasis

The human ADAMTS (a disintegrin and metalloproteinase with thrombospondin-like motifs) family of 19 secreted, multidomain proteolytic enzymes is involved in a wide range of biological processes including ECM assembly and degradation, hemostasis, organogenesis and the regulation of angiogenesis. Defects in certain family members give rise to inherited human genetic diseases, while aberrant expression of other ADAMTSs has been linked to the pathogenesis of arthritis and cancer. Several ADAMTSs act as tumor or metastasis suppressors whose functions are lost either by mutation or epigenetic silencing during tumor progression. This review looks in depth at the involvement of ADAMTSs as positive and negative mediators in cancer growth and spread

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

Abstract Extracellular vesicles (EVs) are lipid‐membrane nanoparticles that are shed or secreted by many different cell types. The EV research community has rapidly expanded in recent years and is 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

Characterization of a rationally engineered nitric oxide, nitrate and nitrite biosensor linked to a hybrid bacterial-­mammalian promoter

<p>Synthetic biology is principally concerned with the rational design and engineering of biological systems that serve useful applied purposes. Biosensors are of particular interest to the field since they serve a broad array of applications, such as medical devices, environmental sensors for the detection of contaminants, toxins or pathogens or in metabolic engineering, to monitor product formation. In this study, we describe the characterization of a family of four nitric oxide, nitrate and nitrite whole­cell biosensors that are based upon a hybrid bacterial­-mammalian promoter design. The hybrid­ design of the synthetic promoter has been engineered for the detection of these nitrogenous species across both bacterial (Escherichia coli) and mammalian systems (MCF­-7). As such, these biosensors may be useful across applications as diverse as cancer therapeutics and the agricultural monitoring of nitrates and nitrites in fertiliser­ treated soil. Qualitative and quantitative analysis of these biosensors in E. coli confirmed that all four biosensor designs (termed B­M_eCFP, B­M_mRFP, M­B_eCFP and M­B_mRFP) were able to quantitatively detect 5-­20 mM of potassium nitrate. In summary, these pilot data suggest that, with further characterisation, this family of biosensors will be able to assess nitrogenous species present within both bacterial (E. coli) and mammalian systems (MCF­7).</p

EcoFlex: A Multifunctional MoClo Kit for <i>E. coli</i> 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 optimization, 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 <i>Escherichia coli</i> has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in <i>E. coli</i>, 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), characterize <i>in vivo</i> and <i>in vitro</i> 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 minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in <i>E. coli</i> synthetic biology

Flow cytometry analysis of P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i> from waste-media cultures.

<p><i>E</i>. <i>coli</i> MG1655 transformed with either empty vector [EV], native [N], constitutive [C] or hybrid [H] <i>phaCAB</i> constructs were cultured in 5 ml of waste-media for 36 h at 37°C. P(3HB) content was assessed via flow cytometry analysis of Nile Red staining. (<b>A</b>) Representative forward scatter (FSC) and side scatter (SSC) contour plots. (<b>B</b>) Representative histogram (FL-5). (<b>C</b>) Normalized fluorescence of Nile Red stained <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, from three independent experiments. Error bars, +/- the standard deviation. Student t-test, *P<0.05 and ***P <0.001.</p

P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>.

<p><i>E</i>. <i>coli</i> MG1655 transformed with empty vector, native, constitutive or hybrid <i>phaCAB</i> constructs were cultured in 1 liter LB media, supplemented with 3% glucose (w/v) for 24 hours or 48 hours. P(3HB) was purified from these cultures and measured as <b>(A)</b> P(3HB) production (g/L) and <b>(B)</b> P(3HB) content (weight [wt.] % of cell dry weight [CDW]). Data represent the mean +/- the standard deviation of three independent experiments. Student t-test, *P<0.05, **P <0.01, ***P <0.001 and ****P <0.0001.</p