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WELCOME
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
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
Dr Paul Race
School of Biochemistry, University of Bristol
Kill or cure: Repurposing bacterial adhesins for therapeutic payload delivery
Abstract: Adherence of bacteria to biotic or abiotic surfaces is a prerequisite for host colonisation and represents an important step in microbial pathogenicity. This attachment is facilitated by bacterial adhesins at the cell surface. Due to their size and often elaborate multi-domain architectures, these polypeptides represent challenging targets for detailed structural and functional characterisation. Here I outline how fundamental studies of the multifunctional fibrillar adhesin CshA have revealed a hitherto unreported mechanism of human cell surface binding, which is now being exploited as the basis for a stem cell homing technology. Biography: Paul is a senior lecturer in Biochemistry at the University of Bristol. He leads the pan-UK EPSRC funded Manufacturing Immortally project, which aims to develop bio-hybrid self-healing ‘living materials’. He is a former Co- Director of the BrisSynBio synthetic biology research centre, and was a founding Director of the Bristol BioDesign Institute. He is co-founder and non-executive Director of the University of Bristol spinout company Zentraxa Ltd., which uses a proprietary biodesign platform to develop bioadhesives for aerospace, marine and medical applications.
PLENARY SPEAKERS
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
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: Opening session
On-demand gene expression patterning and signalling pathway activity in
mammalian cells
Elisa Pedone, Dan Rocca, Lorena Postiglione
Engineering Mathematics Department, School of Cellular and Molecular Medicine and BrisSynBio, University of Bristol ABSTRACT Cellular function, including cell-decision making is orchestrated by the dynamic interplay between signalling pathway activity and the extracellular environment, invariably giving rise to highly complex gene expression patterns. Consequently, to fully understand gene function, the link between dynamic behaviour and cellular phenotype, and to direct cell fate in a user-defined way, tools enabling not only gene overexpression or silencing, but also precise tuning of gene activation levels and temporal dynamics are required. Here, we applied an array of synthetic biology tools to engineer in real-time, on demand gene expression dynamic profiles and signalling pathway activity in mammalian cells. Moreover, exploiting a microfluidics/microscopy platform, harnessing both feedback control (i.e. Model Predictive Control) and segmentation algorithms, we enable robust and real-time regulation of both exogenous and endogenous processes in mammalian cell lines, including embryonic stem cells. Moreover, we show that the experimental and computational platform allows the precise engineering of arbitrary gene expression dynamics in mammalian cells by interfacing biological systems with virtual in silico counterparts. Our methodology can be applied for precisely dissecting threshold effects in gene regulation, unraveling the role of complex gene expression patterns in mammalian cell fate decisions, and for the rapid prototyping of synthetic circuits.
ORAL PRESENTATIONS: Session 1: Design
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
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 2: Medical applications
vSAGE: self-assembled peptide cages presenting immunogenic peptides and
proteins as a vaccine delivery system
Caroline Morris
School of Chemistry and BrisSynBio, University of Bristol ABSTRACT Vaccines have been pivotal to the development of modern medicine, from general childhood vaccination to the global eradication of smallpox and near elimination of polio. Despite this, vaccine development is still an important area of research. Recently, we reported the design, construction and characterisation of self-assembling peptide cages (SAGEs). SAGEs comprise two complementary building blocks, termed hubs, made of de novo homotrimeric and heterodimeric α-helical peptides. Mixing of hubs in aqueous solution leads to co- assembly, first into hexagonal lattices and then into particles approximately 100 nm in diameter. SAGEs can act as readily-accessible synthetic peptide scaffolds for dose-dependent delivery of immunogenic components, capable of inducing and boosting a specific and tailored immune response. Proof of concept work to date has shown that SAGEs functionalised with model antigenic peptides are capable of driving antigen-specific responses both in vitro and in vivo , demonstrating the potential of the system to act as a modular scaffold for vaccine delivery.
ORAL PRESENTATIONS: Session 2: Medical applications
Fabricating an extracellular matrix analogue using natural polymers for cartilage
tissue engineering applications
Runa Begum
School of Cellular and Molecular Medicine, University of Bristol ABSTRACT Osteoarthritis is the most common degenerative disease of the joint. Current treatment options prove inadequate at restoring full joint function due to the complexity of the implicated cartilage tissue. This avascular connective tissue consists of a specialised extracellular matrix (ECM) with a nanofibre-based hierarchical ultrastructure that imparts remarkable biomechanical properties to the tissue; essential for protecting the articular surfaces of load-bearing joints. The advent of smart biomaterials in the field of tissue engineering offers hope to alleviate the shortcomings in current treatments as they can better recapitulate the natural structure of cartilage ECM. We have demonstrated previously that blends of cellulose and silk can stimulate human bone marrow stem cell (BMSC) chondrogenesis without biochemical induction^1. An electrospinning technique has been employed to fabricate nanofibrous materials that can mimic the in vivo ECM using these blends. Cellulose and silk natural polymers were used to fabricate nonwoven neat and composite nanofibres. Scanning electron microscopy was used to characterise the materials. Fibre diameters could be tuned through adjustments in electrospinning flow rate and voltage. Further, environmental temperature and humidity influenced resulting fibre diameters and beading. Fourier transform infrared spectroscopy confirmed the absence of solvent following regeneration of the polymer materials. Neat cellulose, neat silk and composite cellulose:silk in 50: and 75:25 mass ratios were fabricated to produce nanofibrous 3D scaffolds, and fibre diameters could be systematically tuned in the range of 150 to 60 nanometres. All materials demonstrated biocompatibility when cultured in vitro with BMSCs over a 14 day study period. Accordingly, the biocompatible neat and composite natural polymer nanofibres show promise for tissue engineering applications. Work is currently underway to explore the inductive capability of this configuration in driving stem cell differentiation, specifically chondrogenic gene expression^1. References [1] N. Singh, S. S. Rahatekar, W. Kafienah et al “Directing chondrogenesis of stem cells with specific blends of cellulose and silk” Biomacro. 2013.
ORAL PRESENTATIONS: Session 3: Systems
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
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
1. Baculovirus-delivered CRISPR toolkits for homology-independent gene
replacement
Francesco Aulicino 1,2, Charles Grummitt1,2, Julien Capin^1 , Christiane Berger-Schaffitzel1,2, Mark Dillingham1,2^ and Imre Berger1,
- (^) University of Bristol, School of Biochemistry, Biomedical Sciences Building, University Walk, Clifton BS8 1TD
- (^) BrisSynBio, University of Bristol, Life Sciences Building, Tyndall Avenue Bristol, BS8 1TQ ABSTRACT We have developed a novel NHEJ-based DNA-editing strategy which allows to excise a stretch of endogenous DNA and replace it with a synthetic sequence with base-pair precision. We named this approach HITR for Homology-Independent Targeted gene Replacement. HITR will expand CRISPR applications by allowing reliable genome re-writing, combining the efficiency of a NHEJ-based repair approach (editing in dividing and non-dividing cells) with an HDR-like gene-editing outcome (gene replacement). We will explore “helper” strategies to maximise HITR efficiency including genetically encoded modulators of NHEJ activity and DNA tethering strategies aimed at increasing the local concentration of the donor DNA template at the edited site. Delivery of multiple components is an outstanding challenge for currently available delivery systems and impairs the efficiency of any gene-editing approach. For the same reason, exploration of genetically encoded helper strategies for precise gene editing has not been attempted to date. We will therefore repurpose Baculovirus expression vectors (BEVs) for the delivery of CRISPR-based HITR gene-editing toolkits. Thanks to their extensive cargo capacity, safety and delivery efficiency, BEVs will accommodate HITR toolkits and multiple helper modules in a single all-in-one vector.
POSTER PRESENTATIONS
2. Tuneable genetic devices
Vittorio Bartoli^1 , Mario di Bernardo^1 , Thomas E. Gorochowski^2 (^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.
