BrisSynBio Conference: Gene Expression, Signalling Pathways, and Synthetic Biology, Study notes of Biology

Download BrisSynBio Conference: Gene Expression, Signalling Pathways, and Synthetic Biology and more Study notes Biology in PDF only on Docsity! WELCOME 1 Bristol BioDesign Institute Biomolecules to biosystems from understanding to design The Bristol BioDesign Institute (BBI) is one of the University of Bristol’s Specialist Research Institutes. The BBI brings together BrisSynBio, a UK Synthetic Biology Research Centre, the SynBio Centre for Doctoral Training, our Innovation Programme and Public Engagement activities. With wide-ranging applications from health to food security, BBI combines pioneering synthetic biology approaches with understanding biomolecular systems to deliver the rational design and engineering of biological systems for useful purposes. This is delivered through multidisciplinary research which brings together postgraduate and postdoctoral researchers, academics, policy makers and industry, whilst also engaging the public with emerging solutions to global challenges. The BBI places the University of Bristol among the forerunners of UK and international synthetic biology and biodesign research, teaching and innovation. Director: Professor Dek Woolfson Co-Directors: Professor Imre Berger Professor Claire Grierson Professor Mario di Bernardo Conference organising committee: Imre Berger, BrisSynBio Director and Bristol BioDesign Institute Co-Director Graham Day, PhD Student, School of Cellular and Molecular Medicine Bethany Hickton, PhD student, School of Cellular and Molecular Medicine Kathleen Sedgley, Bristol BioDesign Institute Manager Mark Winfield, Post-doctoral Research Assistant, School of Biological Sciences Marie Woods, Bristol BioDesign Administrator Dek Woolfson, BrisSynBio PI and Bristol BioDesign Institute Director Ioannis Zampetakis, PhD Student, Department of Aerospace Engineering Image: Claudia Stoker, Vivid Biology CONTENTS 2 Contents Bristol BioDesign Institute................................................................................................................... 1 PLENARY PRESENTATIONS Dr Paul Race School of Biochemistry, University of Bristol ................................................................ 4 Professor Katharina Landfester Max Planck Institute for Polymer Research, Mainz, Germany ........ 5 PUBLIC LECTURE Professor Nadrian Seeman New York University ................................................................................ 6 ORAL PRESENTATIONS On-demand gene expression patterning and signalling pathway activity in mammalian cells .......... 8 Designing genomes using a computational design-build-test cycle ................................................... 9 Protein design in the cell: De novo bacterial cytoscaffolds .............................................................. 10 How molecular modelling can support synthetic biology, and vice versa ........................................ 11 Investigating lanthanide binding to designed coiled coil trimers ..................................................... 12 vSAGE: self-assembled peptide cages presenting immunogenic peptides and proteins as a vaccine delivery system ................................................................................................................................. 13 Fabricating an extracellular matrix analogue using natural polymers for cartilage tissue engineering applications ................................................................................................................... 14 Using bacterial adhesins to direct human stem cells to the myocardium........................................ 15 Out of equilibrium protocell systems ............................................................................................... 16 Molecular membrane engineering for nanoreactors ....................................................................... 17 POSTER PRESENTATIONS 18 Poster abstracts…………………………………………………………………………………………………………………….19 - 53 PLENARY SPEAKERS 5 Professor Katharina Landfester Max Planck Institute for Polymer Research, Mainz, Germany Nanocapsules as cell modules Abstract: Since many years, there is a quest for minimal cells in the field of synthetic biology, potentially allowing a maximum of efficien¬cy in biotechnological processes. Although the so-called “protocells” are usually referred to in all papers that attempt a cumulative definition of Synthetic Biology, research in this area has been largely underrepresented. Our aim is at developing vesicular structures, i.e. protocells, based on block copolymer self-assembly and engulfed nanocontainers with incorporated functions, such as energy production and the control of transport properties through nanomembranes. Therefore, we have designed and developed nanocapsules that act as cell-like compartments and can be loaded with enzymes for synthetic biology and chemistry. In addition, self- assembly of well-defined diblock copolymers has been used to generate polymersomes and hybrid liposomes/polymersomes. Both strategies allow the compartimentalization on the nano- or microscale and conducting enzymatic or chemical reactions in the confinement of the polymersomes/ nanocarriers. New block copolymers and permeable nanocarriers have been synthesized and optimized. With these protocols we were able to establish an enzymatic reaction cascade within droplet-based compartments. These compartments can act as cell-like functions to regenerate NAD. For these tasks, novel conductive polymer nanoparticles have been developed which will be included into the protocells for the NAD regeneration by light. Also enzyme-complexes are assembled that will fulfill these requirements. Transmembrane transport of ions, molecules and particles is also fundamental to functionality in biology. However, the direct investigation in living cells is very difficult due to the complexity of biological membranes and the diverse coupling of interactions. Therefore, transport of nanoparticles into a minimal model system, based also on a vesicle-forming amphiphilic copolymer was probed in our group. The physical properties of these copolymer molecules are similar to phospholipids and therefore provide the necessary fluidity of a membrane, while ensuring excellent mechanical stability at the same time. The latter is due to the slow exchange of polymer chains between aggregates compared to the experimental time scale (kinetically trapped or “frozen” structures). In addition, the use of the synthetic membrane allows the uncoupling of all involved interactions and processes. Biography: Katharina Landfester received her doctoral degree in Physical Chemistry after working in 1995 at the MPI for Polymer Research (MPIP). After a postdoctoral stay at the Lehigh University (Bethlehem, PA), she worked at the MPI of Colloids and Interfaces in Golm leading the mini-emulsion group. From 2003 to 208, she was professor at the University of Ulm. She joined the Max Planck Society in 2008 as one of the directors of the MPIP. She was awarded the Reimund Stadler prize of the German Chemical Society and the prize of the Dr. Hermann Schnell Foundation, followed by the Bruno Werdelmann Lecturer in 2012 and the Bayer Lecturer in 2014. Her research focusses on creating functional colloids for new material and biomaterial applications. PUBLIC LECTURE 6 Professor Nadrian Seeman New York University DNA: Not merely the secret of life Abstract: We build branched DNA species that can be joined using Watson-Crick base pairing to produce N-connected objects and lattices. We have used ligation to construct DNA topological targets, such as knots, polyhedral catenanes, Borromean rings and a Solomon's knot. Nanorobotics is a key area of application. We have made robust 2-state and 3-state sequence- dependent programmable devices and bipedal walkers. We have constructed 2-dimensional DNA arrays with designed patterns from many different motifs. We have used DNA scaffolding to organize active DNA components. We have used pairs of 2-state devices to capture a variety of different DNA targets. We have constructed a molecular assembly line using a DNA origami layer and three 2-state devices, so that there are eight different states represented by their arrangements. We have demonstrated that all eight products can be built from this system. Recently, we connected the nanoscale with the microscale using DNA origami. We have self-assembled a 3D crystalline array and reported its crystal structure to 4 Å resolution. We can use crystals with two molecules in the crystallographic repeat to control the color of the crystals. Rational design of intermolecular contacts has enabled us to improve crystal resolution to better than 3 Å. We can now do strand displacement in the crystals to change their color, thereby making a 3D-based molecular machine; we can visualize the presence of the machine by X-ray diffraction. The use of DNA to organize other molecules is central to its utility. Earlier, we made 2D checkerboard arrays of metallic nanoparticles, and have now organized gold particles in 3D. Most recently, we have ordered triplex components and a semiconductor within the same lattice. Thus, structural DNA nanotechnology has fulfilled its initial goal of controlling the internal structure of macroscopic constructs in three dimensions. A new era in nanoscale control awaits us. Biography: Nadrian C. Seeman was born in Chicago in 1945. Following a BS in biochemistry from the University of Chicago, he received his Ph.D. in biological crystallography from the University of Pittsburgh in 1970. His postdoctoral training, at Columbia and MIT, emphasized nucleic acid crystallography. He was the first to demonstrate the correctness of Watson-Crick A-U base pairing at atomic resolution. PUBLIC LECTURE 7 He obtained his first independent position at SUNY/Albany, where his frustrations with the macromolecular crystallization experiment led him to the campus pub one day in the fall of 1980. There, he realized that the similarity between 6-arm DNA branched junctions and the flying fish in the periodic array of Escher's 'Depth' might lead to a rational approach to the organization of matter on the nanometer scale, particularly crystallization. Ever since, he has been trying to implement this approach and its spin-offs, such as nanorobotics and the organization of nanoelectronics; since 1988 he has worked at New York University, where he is the Margaret and Herman Sokol Professor of Chemistry. When told in the mid-1980s that he was doing nanotechnology, his response was similar to that of M. Jourdain, the title character of Moliere's Bourgeois Gentilehomme, who was delighted to discover that he had been speaking prose all his life. He was the founding president of the International Society for Nanoscale Science, Computation and Engineering. He has published over 300 papers, and has won the Sidhu Award, the Feynman Prize, the Emerging Technologies Award, the Rozenberg Tulip Award in DNA Computing, the World Technology Network Award in Biotechnology, the NYACS Nichols Medal, the SCC Frontiers of Science Award, the ISNSCE Nanoscience Prize, the Kavli Prize in Nanoscience, the Einstein Professorship of the Chinese Academy of Sciences, a Distinguished Alumnus Award from the University of Pittsburgh, the Jagadish Chandra Bose Triennial Gold Medal and the Benjamin Franklin Medal in Chemistry. He received a Prose Award in Biological Sciences for his 2016 book, Structural DNA Nanotechnology, written during a John Simon Guggenheim Fellowship; he is a Thomson-Reuters Citation Laureate, has been elected a Fellow of the AAAS, the Royal Society of Chemistry and the American Crystallographic Association and has been inducted as a Fellow into the American Academy of Arts and Sciences. ORAL PRESENTATIONS: Session 1: Design 10 Protein design in the cell: De novo bacterial cytoscaffolds Lorna Hodgson School of Chemistry, University of Bristol ABSTRACT Protein design involves the creation of entirely new protein sequences with enhanced or novel functional properties using computational modelling and rational design. A contemporary challenge in protein design is to translate in silico and in vitro studies in vivo to produce de novo-designed proteins that assemble and fold in a controlled manner to form functional proteins and structures in cells. At Bristol and with researchers in Kent and London, we have used rational protein design to construct synthetic scaffolds in bacterial cells. We utilise a three-component system comprising a modified shell protein of a bacterial microcompartment, PduA*, and two complementary de novo heterodimeric coiled coil proteins, CC-Di-A/B. When expressed in E.coli the engineered hybrid system assembles to form a network of filaments that permeate throughout the entire bacterial cytoplasm. Fluorescent proteins and functional enzymes can be specifically targeted and tethered to these intracellular filamentous frameworks through interaction of coiled coil pairs fused to either the PduA* filaments or protein. Furthermore, the scaffold can be directed to the inner membrane of E.coli, coupling synthetic cellular organisation and spatial optimisation through in-cell protein design. These hybrid assemblies can be extracted from cells and therefore can be utilised in both in vivo and in vitro applications as cytoscaffolds and next-generation cell factories, for instance, for the improved production of biofuels in bacteria. We are now attempting to port the cytoscaffolds from bacterial to mammalian cells. ORAL PRESENTATIONS: Session 1: Design 11 How molecular modelling can support synthetic biology, and vice versa Eric J. M. Lang School of Chemistry and BrisSynBio, University of Bristol ABSTRACT Biomolecular modelling and synthetic biology are indispensable to each other. Not only the computational study of engineered biomolecules at an atomistic level of detail can provide crucial information on the designed systems in both predictive and postdictive manners. But the deterministic nature of de novo biomolecules, especially when reduced to a minimal working unit, can also foster the improvement of molecular modelling methods by testing the limits of their accuracy and applicability. Here, we present recent examples on how biomolecular modelling can support synthetic biology and vice versa. First, we describe how molecular dynamics simulations and quantum mechanical calculations helped to shed light on the molecular mechanisms that govern the binding of a fluorescent dye to a set of de novo α-helical barrels (αHBs) and to explain a puzzling induced circular dichroism band observed experimentally. We then detail how the recently developed constant pH molecular dynamics methodology has been used to rationalise the stability of αHBs designed with ionisable residues pointing toward their lumen. Finally, we show how small de novo peptides presenting a highly controlled degree of helicity, can be used to probe the accuracy of protein force field and implicit solvent model combinations. ORAL PRESENTATIONS: Session 1: Design 12 Investigating lanthanide binding to designed coiled coil trimers Oliver J. Daubney School of Chemistry, University of Birmingham ABSTRACT The binding of lanthanides in biological systems has received a great deal of attention for both their photophysical and magnetic properties. Previous work within the group involved the preparation of lanthanide binding coiled coil trimers, however, previous attempts to gain structural information beyond circular dichroism have proven unsuccessful.1,2 Herein we report the design, synthesis and structural characterisation of a family of lanthanide binding coiled coil trimers. The resultant x-ray diffraction results have provided a wealth of structural information regarding the binding site coordination and other external features. The peptides were studied via solution state luminescence and circular dichroism titrations revealing the presence of external binding sites. Interestingly, the metallated complexes were observed by mass spectrometry providing a useful tool to probe the oligomerisation states. DSTL is gratefully acknowledged for a PhD studentship and for the support of this research project through the National PhD Scheme. 1. M. R. Berwick et al., J. Am. Chem. Soc., 2014, 136, 1166-1169. 2. M. R. Berwick et al., Chem. Sci., 2016, 7, 2207-2216. ORAL PRESENTATIONS: Session 2: Medical applications 15 Using bacterial adhesins to direct human stem cells to the myocardium Wenjin Xiao School of Cellular and Molecular Medicine, University of Bristol ABSTRACT The efficient delivery and adherence of cells to a site of interest, a process termed “homing”, remains an elusive goal for cell therapies. Intravenous or intra-arterial infusion of cells inevitably leads to the undesired and detrimental accumulation of the cells at the lungs and liver, which reduces the efficiency of systemic delivery and increases the likelihood of producing lethal microemboli. We have developed a new methodology involving the introduction of exogenous proteins directly anchored to the cell membrane in order to modulate cell behaviour and achieve targeted homing.1 In this work, we showed that a designer protein-surfactant hybrid construct with inherent cardiac tissue homing properties can be rationally engineered to spontaneously insert into the plasma membrane of human mesenchymal stem cells (hMSCs). This was achieved by hijacking the in-built ability of Streptococcus gordonii to home to cardiac tissue by displaying multiple copies of the fibronectin binding domain of the bacterial adhesion protein (bap) 2 on the surface of hMSCs. In order to anchor the construct to the membrane, the bap was fused to supercharged GFP (scGFP), which was then conjugated to surfactant molecules displaying membrane binding properties. The results shown in this work demonstrate that the construct maintains the dual biophysical properties of bap and scGFP, and associates with hMSC membrane with no visual changes in cell morphology. Significantly, the construct is not cytotoxic, does not elicit an hematologic response in mice, and directs hMSCs delivered either intracardially or intravenously to the myocardium, without a concomitant increase in the lungs. This cell membrane display system is completely independent of cell type and can therefore be readily applied to other cell types using a wide array of protein-based targeting molecules. 1. Armstrong, J. P. K. et al. Nat. Commun. 6, 7405 (2015). 2. Back, C. R. et al. J. Biol. Chem. 292, 1538–1549 (2017). ORAL PRESENTATIONS: Session 3: Systems 16 Out of equilibrium protocell systems Liangfei Tian Centre for Protolife Research and Centre for Organized Matter Chemistry and BrisSynBio, School of Chemistry ABSTRACT The potential to develop rudimentary representations of life via the bottom-up construction of functional protocells is an emerging area of synthetic biology. One of the distinguishing feature of living cell is non-equilibrium dynamics, involving constant energy dissipation and kinetic control. By contrast, current research has focused on the exploration of different protocell models at equilibrium states. Thus, the design and construction of protocell communities that can operate at far-from- equilibrium states remains a key challenge. Here, we present a bottom-up approach to study the collective behaviors of ordered arrays of protocells under transient chemical diffusion fields and explore inter-protocell dynamics and environment/protocell interactions as a step towards far from equilibrium protocell systems capable of biochemical sensing and biocomputing. We show that a 2D array of protocells can response spatiotemporally to fluctuating chemical inputs and demonstrate for the first time an example of ‘protocellular differentiation’. Specifically, periodic and dynamic patterns are generated in the 2D protocell arrays by using concentration gradient fields associated with the input of two chemical morphogens. ORAL PRESENTATIONS: Session 3: Systems 17 Molecular membrane engineering for nanoreactors Natalie Di Bartolo School of Biochemistry and BrisSynBio, University of Bristol ABSTRACT This presentation will describe progress in designing, producing, characterising and implementing membrane encapsulated nanoreactors. Our approach is to create energy-transducing vesicles that incorporate engineered membrane proteins and soluble components and to use light to generate electrochemical gradients to power and gate transport of materials between the nanoreactor and its environment. A key aspect of this work is to gain control over the composition of individual nanoreactors and we have explored numerous potential genetically-encodable linking technologies to engineer component membrane proteins that self-assemble into hetero-oligomeric complexes with defined stoichiometry and orientation. SpyCatcher-SpyTag, for example, has been used to assemble a hetero-dimeric XylE-reaction centre complex which can reconstituted into lipid vesicles to create nanoreactors capable of light-driven sugar transport. The impact on reconstitution and sidedness in assembled vesicles is being studied through the development of simple and convenient assays based on the His-tags carried by the component membrane proteins. Digital holographic microscopy, which measures refractive index changes, is being developed as a non-invasive measurement of transport in a range of membrane systems including giant unilamellar vesicles. When coupled with conventional spectroscopic techniques, this will allow us to engineer nanoreactors with optimised flux of reactants and products in response to an applied stimulus. POSTER PRESENTATIONS 20 2. Tuneable genetic devices Vittorio Bartoli1, Mario di Bernardo1, Thomas E. Gorochowski2 1 Department of Enguneering Maths and 2 School of Biological Sciences, University of Bristol ABSTRACT Synthetic genetic circuits regulate gene expression to perform biological computations and control cellular behaviours. These are generally built from genetic devices where the input-output relationship (response function) is assumed to be robust. By ensuring that the responses of connected parts are compatible, a desired overall function can be achieved. Unfortunately, factors like retroactivity, changes in cellular state, and differing environments can significantly alter the behaviour of individual parts and lead to a breakdown in function. Here, we present several novel genetic devices (a sensor and a NOT-gate) which allow for transcription and translation to be separately “tuned” to enable their response function to be dynamically altered. We experimentally characterise the function of these devices, derive their mathematical models to support future in silico design, and use particle swarm optimisation for their parameterisation. Being able to tune the behaviour of genetic devices within a circuit offers a means to create more robust circuits that can adapt to changes over time. Such capabilities will be essential to tackle real-world applications where genetic circuits must reliably function in the face of significant variability. POSTER PRESENTATIONS 21 3. Designing SAGE nanoparticles for endosomal escape Holly Baum 1,2, Lorna Hodgson 1, Joe Beesley 3, Paul Verkade 1, George Banting 1, Dek Woolfson 2, 3 1 School of Biochemistry, Biomedical Sciences Building, University Walk, Bristol, UK 2 BrisSynBio, University of Bristol 3 School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom ABSTRACT Encapsulated drug delivery systems have the potential to overcome many of the drawbacks of conventional therapies, such as toxic side-effects and drug instability. Here we are investigating the use of de novo designed self-assembling cage-like particles (SAGEs) for the delivery of bioactive molecules into cells. SAGEs have the potential to be ideal delivery vehicles due to their modular, adjustable, peptide components and non-cytotoxic nature. In mammalian cell culture SAGEs are predominantly endocytosed through dynamin-dependent pathways, with TEM imaging suggesting caveolae to be the major route of entry. The rate of endocytosis can be finely controlled through modification of the SAGE surface with either positively- or negatively- charged peptides, and this can be quantified by flow cytometry. Internalised SAGEs traffic through early and late endosomes to a final fate of lysosomal degradation, with an intracellular half-life of approximately 3 hours. To avoid this degradative fate, and deliver intact cargo into the cytosol, several pH-responsive design strategies have been employed. A quantitative assay has been developed to detect endosomal escape, and preliminary data using these two designs is encouraging. POSTER PRESENTATIONS 22 4. Investigating enzyme dynamics using simulations – the role of heat capacity in catalysis Michael S Connolly1, Marc W Van der Kamp1, Erica J Prentice2, Vickery L Arcus2 and Adrian J Mulholland1 1School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, United Kingdom 2School of Science, University of Waikato, Hamilton 3240, New Zealand ABSTRACT Enzymes are efficient catalysts that facilitate chemical reactions in biological processes. They are vital for life and play a large role in combatting or causing many diseases in humans, and are also potentially useful as industrial catalysts. Despite this, the origins of enzyme chemistry are still relatively poorly understood. Recent work attempts to understand the role of dynamics in enzyme catalysis by studying the change in heat capacity of activation. This unique view suggests that enzyme dynamics play a large role in their catalytic properties, and could provide an explanation as to why enzymes are such large proteins, why they have such complex and varied structures, and how the optimum temperature of each enzyme is “tuned” by evolution. This has been termed macromolecular rate theory (MMRT), and provides a theoretical justification of the curvature of enzyme rate profiles. By measuring thermodynamic properties of enzyme-catalyzed reactions using classical molecular dynamics (MD) simulations it may be possible to identify and investigate the physical origins of enzyme catalysis. This could provide a route to designing better biocatalysts for use in chemical reactions in industrial processes and help explain how organisms evolve to live at different temperatures. POSTER PRESENTATIONS 25 7. Designing a biocompatible and catalytic de novo membrane protein Christophe J. Lalaurie1, Virginie Dufour1,3, Anna Meletiou1, Sarah Ratcliffe1, Abigail Harland1, Olivia Wilson1, Chiratchaya Vamasiri1, Deborah K. Shoemark1,3, Christopher Williams2,3, Richard B. Sessions1,3, Matthew P. Crump2,3, J.L Ross Anderson1,3 and Paul Curnow1,3* 1 School of Biochemistry and 2 School of Chemistry, University of Bristol, UK. 3 BrisSynBio, Life Sciences Building, Tyndall Avenue, Bristol, UK. ABSTRACT Membrane proteins account for 20% of all natural proteins and are responsible for an array of critical biological processes. The design of membrane proteins from first principles (de novo) is thus attractive as a way to emulate and engineer cellular systems. However, most of the previous work in this field has studied chemically-synthesised peptides in artificial lipid membranes. To bridge the gap between synthetic components and organismal biology we present a genetically-encoded, de novo integral membrane protein that can be recombinantly expressed by Escherichia coli. This artificial sequence is localized to the bacterial cytoplasmic membrane with the intended transmembrane topology and can be extracted in detergent micelles for characterisation. REAMP is robust and malleable, and can be engineered to coordinate heme with properties reminiscent of natural b-type cytochromes. Remarkably, this de novo membrane hemoprotein is capable of nascent redox catalysis. We have since used bioinspired design to develop a second generation protein – REAMP2.0 – with improved properties. Collectively, these results pave the way for the cellular integration of abiotic membrane sequences and show that cellular biosynthesis is a viable alternative to synthetic chemistry for the production of functional de novo membrane proteins. POSTER PRESENTATIONS 26 8. Supercharged enzyme-polymer surfactant nanoclusters for the preparation of smart materials for the decontamination of organophosphorous compounds Graham J. Day1, Andrew Collins2, Mark Sambrook3, Adam W. Perriman1 1 School of Cellular and Molecular Medicine, University of Bristol 2 Quantum Engineering Technology Labs, University of Bristol 3 Defense Science and Technology Laboratory, Porton Down ABSTRACT Organophosphorus nerve agents (OPs) are highly neurotoxic compounds that irreversibly inhibit the enzyme acetylcholinesterase in the nervous system, leading to vomiting, suffocation, and a painful death. However, the use of organophosphorus pesticides has driven the evolution of phosphotriesterase enzymes in soil bacteria that hydrolyse OPs with remarkable efficiency. Exploitation of these enzymes offers an attractive opportunity to synthesise novel materials to decontaminate OPs in the environment. Here, we describe the design of a modular fusion construct comprising two proteins: a phosphotriesterase and a supercationic green fluorescent protein. The solvent-exposed cationic residues provide anchor points for the electrostatic coupling of anionic surfactant molecules, which drive the self-assembly of dense clusters, which can be crosslinked to yield enzymatically active high surface area films. Furthermore, we show these clusters can assemble on cotton fibre templates and be crosslinked in situ to produce similarly active nanocomposite materials. The rational design of these modular enzyme-surfactant constructs permits rapid substitution of the phosphotriesterase for any desired variant to target specific OPs. POSTER PRESENTATIONS 27 9. De novo designed coiled coils as protein-protein interaction domains in E. coli Caitlin L. Edgell1, 4, Nigel J. Savery1, 3, Dek N. Woolfson1, 2, 3 [1] Department of Biochemistry, University of Bristol; [2] Department of Chemistry, University of Bristol; [3] BrisSynBio; [4] SynBio CDT. ABSTRACT: Coiled coils are simple protein folds that are frequently found as protein-protein interaction domains in nature. Their sequence-to-structure relationships have been characterised extensively and, as such, there are many examples of de novo coiled coils, designed using the guidelines established through studying natural proteins. However, while many of these novel coiled coils are well characterised in vitro, their behaviour inside cells is often overlooked. By using designed coiled coils as protein-protein interaction domains in artificial transcription factor systems, we can study how novel sequences behave in a complex cellular environment. We show that novel heterotetrameric coiled coils behave as designed both in vitro and in vivo. POSTER PRESENTATIONS 30 12. Nanoscale homing vectors for regenerative engineering Corrigan Hicks1, 2, Wenjin Xiao1, Costanza Emanueli3 and Adam W. Perriman1 1. The School of Cellular and Molecular Medicine, University of Bristol, BS8 1TD 2. The Bristol Centre for Functional Nanomaterials, University of Bristol, BS8 1TL 3. The School of Clinical Sciences, University of Bristol, BS2 8DZ ABSTRACT Despite the continual improvements in treatments and outcomes following myocardial infarction (MI), cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Stem cell therapies have been investigated for the replacement and restoration of damaged cardiac tissue but emerging evidence suggests that the stem cells do not persist at the damaged site for very long. It has instead been suggested that myocardial repair is stimulated by paracrine factors secreted by stem cells1. Through rational design of a cell membrane binding protein-polymer conjugate, we observe anchoring of these nanoconstructs to the cell surface resulting in an increase in adhesion of human mesenchymal stem cells (hMSCs) to target tissue. This construct consists of a positively charged fluorescent protein (scGFP) covalently linked to a bacterial adhesin from S. gordonii (CshA), which binds to fibronectin (Fn) and allows recognition of and direction towards damaged cardiac tissue2. Here we look to apply this technology towards cell-free therapies, with the construction of surface-modified exosomes for in vivo cardiovascular repair. Exosomes are extracellular vesicles (30 -100 nm in diameter) which have been shown to promote angiogenesis and improve blood-flow to damaged cardiac tissue in mice and large animals with both acute and chronic heart disease3. By combining these properties with the aforementioned homing ability, the nanoconstruct-modified exosomes have the potential to have a serious impact in regenerative engineering. References 1. De Jong, O. G., Van Balkom, B. W. M., Schiffelers, R. M., Bouten, C. V. C. & Verhaar, M. C. Extracellular Vesicles: Potential Roles in Regenerative Medicine. Front. Immunol. 5, 608 (2014). 2. Armstrong, J. P. K. et al. Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue. Nat. Commun. 6, 1–6 (2015). 3. Beltrami, C. et al. Human pericardial fluid contains exosomes enriched with cardiovascular- expressed microRNAs and promotes therapeutic angiogenesis. Mol. Ther. 25, 679–693 (2017). POSTER PRESENTATIONS 31 13. Stimuli-responsive protocells: a synthetic approach Mary Jenkinson-Finch, Pierangelo Gobbo, Stephen Mann*. Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry University of Bristol, Bristol, BS8 1TS (UK) ABSTRACT Compartmentalised cell-like entities (protocells) have been constructed from a wide range of materials and have gained much interest in the scientific community as a means of modelling complex biological systems, and of advancing future living technologies. In particular, proteinosomes are self- assembling membrane-bound microscale constructs which have been shown to be capable of biomaterial encapsulation, selective permeability, gene directed protein synthesis, and collective behaviors. In this presentation we show that the chemical programming of proteinosome membranes can provide an opportunity for new stimuli-responsive protocells and prototissues to be synthesized with unprecedented emergent behaviors, and the possibility of new targeted delivery systems. Here we present two novel proteinosome phenotypes: one which responds directly to UV light, and one which responds to a decrease in pH which can be initiated in situ by an enzyme cascade reaction or via communication between protocell communities. Providing proteinosome communities with programmed chemical reactivity will escalate progression towards practical models of life-like systems, and will advance the development of complex functional microsystems and protocellular ecosystems. POSTER PRESENTATIONS 32 14. Towards novel 3D-printed biomaterials via enzymatic-mediated polymerisations R. George Klemperer, Dr Ross Anderson, Dr Adam Perriman Schools of Biochemistry and Cellular and Molecular Medicine, University of Bristol, and Synthetic Biology CDT ABSTRACT Enzyme-mediated polymerizations have a remarkable efficiency under mild conditions and are attracting a lot of interest due to their environmental friendliness and potential for in situ polymerizations. Apropos of this, a de novo peroxidase enzyme designed in the Anderson lab at the University of Bristol will be expressed in E. coli and then isolated and purified. The enzyme will then be used to mediate the polymerization of acrylamide at physiological pH and temperature as part of a ternary initiation system of the enzyme, along with hydrogen peroxidase and acetylacetone. The polymerization products will then be analysed for yield, viscosity and average molecular weight. The global objective is to incorporate the C45 ternary initiation system and monomer formulation into a bioink, recently developed by the Perriman group at the University of Bristol, so the material (a bioink/polymer hybrid) can be patterned via 3D bioprinting. Here, the reaction components will be incorporated as either the isolated purified components or via direct dispersion of C45-expressing E. coli into the bioink. Following this, more complicated material syntheses will be attempted. Potentially incorporating smart polymers/gels to create materials with feedback properties and embedded intelligence. POSTER PRESENTATIONS 35 17. Engineering the abyssomicin C biosynthetic pathway to produce functionally optimised ‘unnatural’ natural products. Maschio, L1; Marsh, CO1; Parnell, AE1; Han, LC2; Scott, A3; Willis, CL2; Race, PR1. 1 School of Biochemistry, University of Bristol; 2 School of Chemistry, University of Bristol; 3 DSTL, Porton Down. ABSTRACT Abyssomicin C is a broad-spectrum antibacterial natural product that was first isolated from the marine bacterium Verrucosispora maris. This compound exhibits potent bioactivity against methicillin resistant Staphylococcus aureus (MRSA) and shows potential for development as a clinically useful antibiotic. Abyssomicin C is the simplest of the spirotetronate polyketides, and studies of the biosynthetic pathway to this compound have revealed a multitude of enzymatic peculiarities. Here I outline progress across three strands of activity: i) Attempts to reconstitute the entire aby biosynthetic pathway in a range of heterologous hosts to enable fundamental studies of the pathway and the development of an industrially relevant production host; and ii) fundamental studies of the aby pathway enzyme AbyU, a bona fide natural Diels-Alderase, exploring substrate selectivity and the ability of this enzyme to generate ‘non-natural’ spirotetronates, as well as its ability to function as a versatile biocatalyst in industry. POSTER PRESENTATIONS 36 18. Engineerd red blood cells as drug delivery agents and bioreactors Marjolein Meinders1, Debbie Shoemark1, Natalie DiBartolo1, Ashley Toye1,2 1 School of Biochemistry, Biomedical Sciences Building, University Walk, Bristol, UK. 2 NHIR Blood and Transplant Research Unit, University of Bristol. ABSTRACT The red blood cell has a well-characterised life cycle and biophysical properties that make them the ideal vehicle for therapeutic use. Research to date has generally focused on loading mature erythrocytes with drugs or functionalization of the erythrocytes surface, alterations that can be detrimental to the biophysical properties of the erythrocytes. Our approach is to use an ex vivo erythroid culture system that allows us to generate immature erythrocytes (reticulocytes) from either CD34+ cells isolated from peripheral blood or alternatively by using novel human immortalised erythroid cell lines.1 We have shown that these erythroid cells can be manipulated by altering their protein expression during erythroid development to engineer erythrocytes with desirable properties. Mitochondrial neurogastrointestinal enchalomyopathy (MNGIE) is a rare autosomal metabolic disorder caused by thymidine phosphorylase (TYMP) deficiency that has been previously treated successfully by encapsulation into erythrocytes. We have generated reticulocytes containing increased levels of TYMP. However, the majority of the overexpressed protein is degraded during erythroid differentiation. We show that this degradation involves ubiquitination, and the application of ubiquitation inhibitors block TYMP degradation. 1. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Trakarnsanga K et al., Nature Communications 2017 Mar 14;8:14750 POSTER PRESENTATIONS 37 19. How robust is icosahedral symmetry in packings of proteins on spherical shells? M. Mosayebi1,2, D. Shoemark2,3, J. Fletcher4, R. Sessions2,3, N. Linden1, D. N. Woolfson2,4, T. B. Liverpool1,2 1 School of Mathematics, University of Bristol, University Walk, Bristol BS8 1TW, UK BrisSynBio, 2 University of Bristol, Tyndall Avenue, Bristol BS8 1TQ, UK
 3 School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK 4 School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK ABSTRACT The formation of spherical cages from protein building blocks is a remarkable self-assembly process in many natural systems where a small number of elementary building blocks are assembled to build a highly symmetric icosahedral cage. In turn, this has inspired synthetic biologists to design de novo protein cages. We use simple models, on multiple scales, to investigate the self-assembly of a spherical cage, focusing on the regularity of the packing of protein-like objects on the surface. Using building blocks, which are able to pack with icosahedral symmetry, we examine how stable these highly symmetric structures are to perturbations that may arise from the flexibility of the interacting blocks. We find that, in the presence of those perturbations, icosahedral packing is not the most stable arrangements for a wide range of parameters; rather disordered structures are often found on the cage surface. Our results suggest that (i) many designed, or even natural, protein cages may not be regular in the presence of those perturbations, and (ii) that minimizing those flexibilities can be a possible design strategy to obtain regular synthetic cages with full control over their surface properties. POSTER PRESENTATIONS 40 22. Computer simulation as a tool in the development of biosynthetic routes to high value monoterpene compounds Kara E. Ranaghan1, Marc W. van der Kamp1,2, Nicole Leferink3, Nigel S. Scrutton3, and Adrian J. Mulholland1 1 Centre for Computational Chemistry, University of Bristol, Clifton, Bristol, BS8 1TS. 2 School of Biochemistry, University of Bristol, Clifton, Bristol, BS8 1TD. 2 Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street Manchester, M1 7DN ABSTRACT Monoterpenes are produced from a single linear substrate, geranyl diphosphate (Fig. 1), by a group of enzymes called the monoterpene cyclases/synthases (mTC/Ss) that catalyse high-energy cyclisation reactions involving unstable carbocation intermediates. Rational engineering of mTC/Ss is hampered by the lack of correlation between the active site sequence and cyclisation type. Available mutagenesis data to shows that amino acids involved in product outcome are clustered and spatially conserved within the mTC/S family. Three important plasticity regions were investigated, with mutations in two of the regions giving rise to products of premature quenching of the reaction. A LimS variant with mutations in the second region (S454G, C457V, M458I), produced mainly more complex bicyclic products. QM/MM MD simulations reveal that the second cyclisation is not due to compression of the C2-C7 distance in the -terpinyl cation, but is the result of an increased distance between C8 of the -terpinyl cation and two putative bases (W324, H579) located on the other side of the active site, preventing early termination by deprotonation. Such insights into the impact of mutations can only be obtained using integrated experimental and computational approaches, and will aid the design of altered mTC/S activities towards clean monoterpenoid products. POSTER PRESENTATIONS 41 23. Social responsibility and gene editing technologies in wheat research Michael Reinsborough BrisSynBio | University of Bristol | Life Sciences Building | Tyndall Avenue | Bristol | BS8 1TQ & Department of Health & Social Sciences | University of the West of England Room 3L12 | Faculty of Health & Applied Sciences | Frenchay Campus | Bristol | BS16 1QY ABSTRACT In confluence with recent BBSRC funding emphasis on synthetic biology research, the Bristol Cereal Genomics Lab attempts to use CRISPR/deadCas9 to explore SPO11 gene driven recombination events in hexaploidal wheat during meiosis, hoping to augment the endogenous recombination system, and create wheat gene recombination relevant to breeders. This poster presentation looks at existing research in wheat genomics seeking to contextualize BrisSynBio lab work within UK agriculture and the longer history of human wheat relations. As the Bristol Biodesign Institute launches an ambitious scientific program, it can draw from examples from BrisSynBio, where researchers have worked to modify human red blood cells, mitochondria and the recombination machinery of crop plants, while also considering the larger social, philosophical, and societal context and drawing in aesthetic communication forms to encourage public dialogue. POSTER PRESENTATIONS 42 24. 3D bioprinting with a dual-crosslinking bioink Thomas Richardson Bristol Centre for Functional Nanomaterials, School of Cellular and Molecular Medicine, University of Bristol ABSTRACT Leveraging techniques developed in the Perriman Group, this project proposes a new method to generate crosslinked hydrogels for 3D bioprinting. 3D bioprinting is a rapidly emerging field of tissue engineering, whereby layers of cell-laden hydrogel are deposited in a computer controlled sequence to form 3-dimensional constructs. To form these bioinks, researchers have explored a range of different materials, from artificial polymers to naturally occurring ones. Initial experiments focused on using a two component system, made of alginate and Pluronic F127. This bioink is first thermally gelled before being crosslinked using CaCl2. However, it contains no biochemical cues normally found in the extra-cellular matrix (ECM). To remedy this, a novel Fibrinogen/Thrombin-based crosslinking step was added, providing more of a mimic for the ECM. This increased viability in the cell type of interest (conditionally-immortalised glomerular podocytes) from 53% to over 75% after 24hrs, as measured by live-dead staining. POSTER PRESENTATIONS 45 27. Developing environments for the study of signal integration and decision making in Escherichia coli Barbara Shannon; Agostino Guarino; Dan Rocca; Fabio Annunziata; Antoni Matyjaszkiewicz; Gianfranco Fiore; Claire S. Grierson; Lucia Marucci; Nigel J. Savery and Mario di Bernardo. BrisSynBio and University of Bristol ABSTRACT The ability of living organisms to process signals from the environment and adapt accordingly is vital for their survival. Information processing and signal integration allow for the activation of appropriate pathways at the right phase of the organisms’ life. Our previous work has described the implementation of a reference-comparator system within Escherichia coli, which allows the cell to tune the expression of GFP by computing the difference between a quorum sensing molecule (AHL) and a chemical inducer (ITPG). This computation is achieved by molecular titration between an orthogonal σ factor and its cognate anti-σ factor. Florescence assays have indicated that the computation system is accurate and able to dynamically adapt GFP expression to different combinations of the two signals. This system represents a resource for biotechnological applications, where it is useful for gene expression to be regulated by multiple signals, allowing the programming of complex behaviour. Here we describe the development of two additional environments, which will allow for the characterisation and progression of this circuit at the single cell level and in batch culture. We also describe the potential for these comparator networks to be extended across multiple cell populations. POSTER PRESENTATIONS 46 28. Ultrasound-guided bioprinting of hydrogel-encapsulated cells for vascular tissue engineering Jenna M Shapiro 1, Bruce W Drinkwater 2, Adam W Perriman 3, Mike Fraser 1 University of Bristol - Department of Computer Science 1, Department of Mechanical Engineering 2, School of Cellular and Molecular Medicine 3 ABSTRACT The formation of a vascular network in a tissue engineered construct in vitro remains an open problem. Acoustic radiation forces are gaining attention as an attractive method of quickly patterning microparticles, including cells, within a support medium, with little to no cellular damage. Accordingly, we describe how hydrogel-encapsulated cells are ultrasonically patterned with their spatial resolution determined by a combination of initial droplet size distribution and the ultrasonic standing wave pattern. By using a two-phase bioink carrier system, comprised of alginate and Pluronic-F127, the microgels aggregate and are held in place due to the temperature-controlled phase transition of the Pluronic, allowing for the assembly of a larger structure. Human umbilical vein endothelial cells maintained good viability both when cultured in bioink, and when exposed to the ultrasonic field. The linear patterning we demonstrate forms a first step towards the generation of larger, high resolution, 3D cellular structures. Crucially, the resolution of this approach has the potential to approach cellular length scales. This technology will be applied to developing multi-scale vascular networks, to incorporate both a large-diameter vessel to enable perfusion, as well as a microvascular network to promote nutrient and metabolite diffusion when embedded into a secondary tissue engineered structure. This work was undertaken as part of project Interaction Design with Functional Plastics, supported by EPSRC grant EP/M021882/1. POSTER PRESENTATIONS 47 29. Atomistic simulations of self-assembled nanocages (SAGEs) Deborah K. Shoemark1, Richard B. Sessions,1Amaurys Avila-Ibarra1, Jordan Fletcher2, James Ross2, Derek N. Woolfson2 and Simon N. McIntosh-Smith3 1 School of Biochemistry, Medical Sciences Building, University of Bristol, BS8 1TD 2 School of Chemistry, Cantock’s Close, University of Bristol, BS8 1TS 3 Department of Computer Science, Merchant Venturer’s Building ABSTRACT Atomistic molecular modelling and dynamics simulations have provided insights into protein design and the pre and postdictive rationalisation of their experimental behaviour for various projects within BrisSynBio. One such project involved an Archer Leadership Award which enabled us to run atomistic molecular dynamics simulations on the self-assembling nanocage structures (SAGEs) from the Woolfson laboratory. SAGEs are built in the laboratory by mixing two types of hub (acidic and basic) each comprised of six 25 residue peptides. The experimental data are consistent with these objects being hollow spheres around 100 nm in diameter with a different exposure of the N and C termini of the peptides at the SAGE surface1. An idealised model of the simplest possible SAGE particle was constructed from 3720 25-mer peptides and MD simulations performed in explicit solvent. Simulations of the parent SAGE and two derivatives were carried out in the presence of other globular proteins and a range of small molecules. The evolution of SAGE structure over the microsecond timescale was monitored along with the way their interaction with solutes. Both adhesion to, and permeation through, the surface SAGE peptide network by these solutes is observed and discussed. POSTER PRESENTATIONS 50 32. Engineering trans-AT PKS antibiotic gene clusters to deliver bioactive compounds Luoyi Wang, Li-Chen Han, Matt Crump, Paul Race, Chris Willis School of Chemistry and BrisSynBio, University of Bristol ABSTRACT Mupirocin is the active ingredient of the ointment Bactroban marketed by GSK. It is a mixture of pseudomonic acids A, B, and C produced by Pseudomonas fluorescens NCIMB 10586 via trans-AT polyketide synthases (PKSs). Thiomarinols are closely related to mupirocin, being produced by the marine bacterium Pseudoalteromonas spp. SANK 73390 via a very similar biosynthetic gene cluster, with the addition of a non-ribosomal peptide synthase (NRPS)-encoded pyrrothine moiety. Our previous studies on detailed metabolic profiling of mutant strains generated by systematic inactivation of PKS and tailoring genes, along with re-feeding of isolated metabolites to mutant stains have provided us insights into mupirocin biosynthesis. These results suggested the presence of many noncanonical features, including the existence of parallel pathways, diverse oxygenation steps, noncolinear PKS gene order, etc. By rational genetic manipulation and heterologous expression of key enzymes from these biosynthetic pathways, we aim to fully understand how polyketides are generated via sophisticated multiple enzyme architectures, which may significantly aid the development of novel bioactive compounds efficiently in scalable amounts and of new biocatalysts of widespread value. POSTER PRESENTATIONS 51 33. BrisSynBio microcryoprobe 700MHz Christopher Williams and Matt Crump BrisSynBio ABSTRACT I will be giving an overview of the BrisSynBio 700MHz 1.7mm microcryoprobe NMR facility housed at the School of Chemistry, University of Bristol. The system offers the one of the highest 1H sensitivities available in the UK for molecular characterisation, biological NMR and high-throughput NMR. This instrument is fully automated with a chilled autosampler and associated liquid handling robotics for high-throughput sample preparation. For small compounds we can acquire 1H 1D spectra in the nanogram range and acquire standard 2D 1H observe experiments such as 1H-13C HSQC spectra on a few micrograms. In addition we can run the full suite of biomolecular triple resonance experiments for protein structure determination on micrograms quantities. The system is ideal for the characterisation of sample limited molecules e.g isolated natural products, metabolites or proteins/peptides that can only be obtained in minute amounts from cell-free or mammalian systems. POSTER PRESENTATIONS 52 34. Using bacterial adhesins to direct human stem cells to the myocardium W. Xiao 1, T. I. P. Green 1,2, X. Liang 3, G. Perry 4, R. Cuahtecontzi Delint 1,2, M. S. Roberts 3,5, K. le Vay 1,2, H. Wang 3, P. R. Race 6, A. W. Perriman*1 1 School of Cellular and Molecular Medicine, University of Bristol, BS8 1TD, UK. 2 Bristol Centre for Functional Nanomaterials, University of Bristol, BS8 1FD, UK. 3 Therapeutics Research Centre, The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia. 4 Sorbonne Université, Laboratoire d’Electronique et d’Electromagnétisme, L2E, F-75005, Paris, France 5 School of Pharmacy and Medical Science, University of South Australia, Adelaide, SA 5001, Australia. 6 School of Biochemistry, University of Bristol, BS8 1TD, UK. ABSTRACT The efficient delivery and adherence of cells to a site of interest, a process termed “homing”, remains an elusive goal for cell therapies. Intravenous or intra-arterial infusion of cells inevitably leads to the undesired and detrimental accumulation of the cells at the lungs and liver, which reduces the efficiency of systemic delivery and increases the likelihood of producing lethal microemboli. We have developed a new methodology involving the introduction of exogenous proteins directly anchored to the cell membrane in order to modulate cell behaviour and achieve targeted homing.1 In this work, we showed that a designer protein-surfactant hybrid construct with inherent cardiac tissue homing properties can be rationally engineered to spontaneously insert into the plasma membrane of human mesenchymal stem cells (hMSCs). This was achieved by hijacking the in-built ability of Streptococcus gordonii to home to cardiac tissue by displaying multiple copies of the fibronectin binding domain of the bacterial adhesion protein (bap) 2 on the surface of hMSCs. In order to anchor the construct to the membrane, the bap was fused to supercharged GFP (scGFP), which was then conjugated to surfactant molecules displaying membrane binding properties. The results shown in this work demonstrate that the construct maintains the dual biophysical properties of bap and scGFP, and associates with hMSC membrane with no visual changes in cell morphology. Significantly, the construct is not cytotoxic, does not elicit an hematologic response in mice, and directs hMSCs delivered either intracardially or intravenously to the myocardium, without a concomitant increase in the lungs. This cell membrane display system is completely independent of cell type and can therefore be readily applied to other cell types using a wide array of protein-based targeting molecules. 1. Armstrong, J. P. K. et al. Nat. Commun. 6, 7405 (2015). 2. Back, C. R. et al. J. Biol. Chem. 292, 1538–1549 (2017). NOTES 55 NOTES 56