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2010 Annual Conference

Synthetic Components Network

Synthetic Biology from the bottom up

23 – 24 September 2010

Claret Jug Function Room

SCN AC, 23 – 24 September 2010 Welcome

Welcome message Dear all, Welcome to Oulton Hall Hotel, and the 2010 Annual Conference of the Synthetic Components Network (SCN). First of all thank you to everyone who has contributed to the exciting conference programme, which represents the breadth and depth of research and expertise across the Network. We hope that the programme and poster sessions, will provide a real insight into some of the work already going on in the Network, highlight some of the areas where people have started together, and reveal new areas ripe for collaboration. Indeed, our overall objective is to foster communication and collaborations across the Network, but, of course, whether this happens is ultimately up to you! We are also very pleased to welcome the PIs or representatives of three other Research Council-funded Networks in Synthetic Biology. They will each be presenting their networks, demonstrating more broadly what is going on in the UK Synthetic Biology community. We have commissioned a short video to show at our partner science centres Explore at Bristol and ThinkTank and other public platforms. Michael will be capturing footage throughout the conference, I hope you will make him welcome, and that some of you will volunteer to be interviewed. On Friday morning we are running a ‘Sci Foo’ session. This is an open session: any one is welcome to contribute to the agenda, show a slide or two, and discuss topics that have emerged during the course of the conference. In particular early career researchers are encouraged to contribute. Because new interactions and exploring potential collaborations are key, we have left plenty of time for networking and discussion over lunches, dinner, poster sessions, and in the bar! We hope you enjoy conference. Best wishes, Dek Woolfson

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SCN AC, 23 – 24 September 2010 Contents

Table of Contents Welcome from Dek 2 Programme 4 Scientific sessions 1: Towards encapsulated systems: dynamics and movement 6 2: Towards encapsulated systems: sensing, acquisitioning and concentrating 10 3: Sci Foo or emerging topics 14 4: Different perspectives 15 5: Biomolecular components 18 Poster sessions PIs 22 PhDs and PDRAs 27 Delegate list 36 Researcher exchange form 39 Feedback form 41

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SCN AC, 23 – 24 September 2010 Programme

Programme ---------------------------------------------------------------------------------------------------------------- THURSDAY 23rd ---------------------------------------------------------------------------------------------------------------- 09:00 – 12:00 Arrivals and registration

12:00 – 13:00 Lunch

Claret Jug function bar

13:00 – 13:15 Welcome Dek Woolfson, Bristol

Session 1: Towards encapsulated systems 1: dynamics and movement Chair: Richard Berry

13:15 – 13:45 David Leigh, Edinburgh

Making the tiniest machines

13:45 – 14:15 Dafydd Jones, Cardiff

Constructing protein components for synthetic biology using new engineering strategies

14:15 – 14:35 Ewan Main, Queen Mary

Repeat proteins, ideal synthetic components

14:35 – 14:55 David Rusling, Southampton

Modifying nucleic acid nanoconstructs by triple-helix formation?

15:00 – 15:30 Discussion

15:30 – 16:00 Tea Claret Jug function bar (group check in for those arriving on Thursday)

Session 2: Towards encapsulated systems 2: sensing, acquisition and concentrating Chair: Andrew Turberfield

16:00 – 16:30 Tony Cass, Imperial Molecular sensing through conformational change: A design and build approach

16:30 – 17:00 Natalio Krasnogor, Nottingham

Network in Synthetic Biology: SynBioNT

17:00 – 17:20 John Colyer, Leeds

Peptide polymers assemble in response to enzyme catalysed modification

17:20 – 17:40 Jordan Fletcher, Bristol

Towards synthetic cytoskeletons

17:40 – 18:10 Discussion

18:10 – 18:30 Free time

18:30 – 19:30 Drinks & Posters

Oulton Suite

19:30 – 21:00 Dinner

Oulton Suite

21:00 – late Bar

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SCN AC, 23 – 24 September 2010 Programme

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---------------------------------------------------------------------------------------------------------------- FRIDAY 24th

---------------------------------------------------------------------------------------------------------------- 7:00 – 9:00 Breakfast

Session 3: Sci Foo or Emerging Topics (‘anything goes’)

Chair: Dek Woolfson, Bristol 9:00 – 10:30

10:30 – 11:30 Coffee & posters

Claret Jug function bar

Session 4: Different perspectives Chair: Maggie Leggett

11:30 – 12:00 Graham Stafford, Sheffield

Network in Synthetic Biology: MATEs Network

12:00 – 12:30 Matthew Kearnes, Durham Upstream public engagement: Building social intelligence into new and emerging technologies

12:30 – 13:00 Antonis Papachristodoulou, Oxford

Network in Synthetic Biology: RoSBNet

13:00 – 14:15 Lunch Claret Jug function bar (Management Committee Meeting – MC only)

Session 5: Biomolecular components Chair: John Colyer

14:20 – 14:50 Jon Bath, Oxford Direct observation of the stepwise movement of a DNA motor

14:50 – 15:20 Leo Brady, Bristol

Protein assembly by design and semi-design

15:20 – 15:40 Tom Williams, Sussex Aβ-membrane interactions and its relation to Alzheimer’s disease

15:40 – 16:00 Masayoshi Tanaka, Leeds Development of highly regulated, biocompatible, nanowires using tubulated liposomes

16:00 – 16:30 Discussion & close

Refreshments served in Claret Jug function bar

---------------------------------------------------------------------------------------------------------------- Notes: Plenary talks: 25 talk + 5 questions Selected orals: 15 talk + 5 questions Poster session: PIs or senior PDRA to present “general” group posters, PhD students and PDRAs to present research project posters ----------------------------------------------------------------------------------------------------------------

SCN AC, 23 – 24 September 2010 Session 1: Towards encapsulated systems: dynamics and movement

Leigh, David Institution: University of Edinburgh

Oral presentation Title: Making the tiniest machines Key words: Synthetic molecular machines, synthetic molecular motors, catenanes, rotaxanes Abstract ....................................................................................................................................... Over the past few years some of the first examples of synthetic molecular level machines and motors—all be they primitive by biological standards—have been developed.1 These molecules respond to light, chemical and electrical stimuli, inducing motion of interlocked components held together by hydrogen bonding or other weak molecular interactions.2

Perhaps the best way to appreciate the technological potential of controlled molecular-level motion is to recognise that nanomotors and molecular-level machines lie at the heart of every significant biological process. Over billions of years of evolution Nature has not repeatedly chosen this solution for achieving complex task performance without good reason. In stark contrast to biology, none of mankind’s fantastic myriad of present day technologies exploit controlled molecular-level motion in any way at all: every catalyst, every material, every polymer, every pharmaceutical, every chemical reagent, all function exclusively through their static or equilibrium dynamic properties. When we learn how to build artificial structures that can control and exploit molecular level motion, and interface their effects directly with other molecular-level substructures and the outside world, it will potentially impact on every aspect of functional molecule and materials design. An improved understanding of physics and biology will surely follow.

1 For representative examples from the Leigh group, see: M. von Delius, E. M. Geertsema and D. A. Leigh, Nature Chem., 2010, 2, 96; C.-F. Lee, D. A. Leigh, R. G. Pritchard, D. Schultz, S. J. Teat, G. A. Timco and R. E. P. Winpenny, Nature, 2009, 458, 314; V. Serreli, C.-F. Lee, E. R. Kay and D. A. Leigh, Nature, 2007, 445, 523; J. Berná, D. A. Leigh, M. Lubomska, S. M. Mendoza, E. M. Pérez, P. Rudolf, G. Teobaldi and F. Zerbetto, Nature Mater., 2005, 4, 704; V. Hernandez, E. R. Kay and D. A. Leigh, Science, 2004, 306, 1532; D. A. Leigh, J. K. Y. Wong, F. Dehez and F. Zerbetto, Nature, 2003, 424, 174. 2 For a review, see: E. R. Kay, D. A. Leigh and F. Zerbetto, Angew. Chem. Int. Ed., 2007, 46, 72. .......................................................................................................................................

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Jones, Dafydd Institution: University of Cardiff

Oral presentation Title: Constructing protein components for synthetic biology using new engineering strategies Key words: Abstract ....................................................................................................................................... Central to synthetic biology is the ability to construct new protein components with desired functionality that may not exist in the natural protein repertoire. Standard protein engineering approaches have already proved useful in constructing simple derivatives of existing proteins for use in synthetic biology and bionanotechnology. For example, the introduction of cysteine residues at strategic positions facilitates binding of proteins in defined orientations to gold substrates for use as single molecule devices. To introduce radically new functionality into existing protein templates, protein engineering approaches are required that sample normally avoided sequence space or even new chemistry. We have employed in vitro evolution to access mutational space that alters the protein backbone, which is normally considered “disruptive” to a protein but opens up new conformational and hence functional space not sampled by simple substitutions alone. By taking an evolutionary approach libraries of proteins can be generated from which variants with the required properties are selected. For example, new protein scaffolds can be constructed that can act as protein switches by marrying the activities of normally disparate proteins. The ability to construct artificial protein switches that change their properties in response to a desired signal allow significant new possibilities for creating novel cellular modulators and biosensors. Targeted proteins can also be evolved to incorporate unnatural amino acids using a modified cellular protein synthesis system. The new chemistry inherent in the unnatural amino acid will allow proteins to sample physicochemical properties not available from within the 20 natural amino acid set and will thus allow the introduction of distinct new functionality.

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Main, Ewan Institution: Queen Mary University London

Oral presentation – SCN Researcher Exchange report Title: Probing the thermodynamic characteristics of novel synthetic proteins Key words: Abstract ....................................................................................................................................... Recently, Main’s group has produced a series of designed TPR and beta helix proteins. In order to further characterise their suitability for biomaterials and novel antibody-like interaction motifs, a study was proposed investigating the biophysical attributes of these repeats as they increase is size through addition of repeat units. In particular, stability and cooperativity of folding was probed using DSC (Differential Scanning Calorimetry) and folding and dynamics explored using ultra fast flow cells. This researcher exchange has yielded a high quality dataset and is being used as part of a paper that will discuss how CTPR proteins can be engineered with a thermodynamic switch for fibril formation. Plans for future studies include investigating the construction of ultra fast mixing cells.

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Rusling, David Institution: University of Southampton

Oral presentation Title: Modifying nucleic acid nanoconstructs by triple-helix formation? Key words: Self-assembly, DNA recognition, Triple-helical nucleic acids Abstract ....................................................................................................................................... The phenomenon of biological self-assembly has been exploited widely in the field of bionanotechnology. One of the forerunners in this field is nucleic acids due to their strong predilection for adopting Watson-Crick base pairing. Hundreds of structures have been constructed by combining synthetic or natural nucleic acid strands ‘programmed’ with base sequences capable of branching or stitching double-helical regions together. By their very nature, the duplex regions within these structures will be capable of interacting with proteins that can make, break or repair DNA. Other methods of DNA and RNA recognition might also be useful in the modification of such structures.

The triplex approach to DNA recognition has so far been developed as a means of modulating the expression of specific genes, with implications in synthetic biology. Triplexes are generated when a third nucleic acid strand binds within the duplex major groove by Hoogsteen hydrogen bonding to exposed groups on the base pairs, generating base triplets. A simple recognition code exists but has also been extended by using synthetic oligonucleotides containing non-natural nucleotides1. The generation of these structures should allow the selective recognition of different regions within a DNA or RNA nanoconstruct. Suitably tagging the third strand with proteins, chemical or functional groups will allow their incorporation at these precise locations. The potential of this approach - with a view to establishing collaborations within the network - will be discussed here.

1Rusling et al. (2005) Nucleic Acids Res. 33; 3025-32. .......................................................................................................................................

SCN AC, 23 – 24 September 2010 Session 2: Towards encapsulated systems: sensing acquisitioning and concentrating

Cass, Tony Institution: Imperial College London

Oral presentation Title: Molecular sensing through conformational change: A design and build approach

Key words: PBP’s, aptamers, electrochemistry, fluorescence Abstract ....................................................................................................................................... The use of molecular flexibility in biology to sense and control cellular processes is a very familiar one and has been tuned through evolution to span a range of amplitudes and timescales depending upon the input (stimulus) and output (response) events.

We have sought to similarly use conformational change to provide new sensing modes with both proteins and nucleic acids that can function as components of analytical devices. Unlike biological outputs where the conformational change is often coupled to changes in molecular interactions; our device based structure switching must induce an alteration in some physical effect that can generate an electrical signal.

To illustrate these ideas we have used large-scale domain motions in periplasmic binding proteins to sense ligands through changes in fluorescence intensity. A second example is in nucleic acid aptamers where binding of a target molecule brings about a structural reorganization that modulates the current-voltage curve at an electrode.

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Krasnogor, Natalio Institution: University of Nottingham

Oral presentation – Network in Synthetic Biology Title: SynBioNT

Key words: Abstract ....................................................................................................................................... The overarching aim of this network is to generate new vigorous interactions between the disciplines that impinge (and contribute to) Synthetic Biology by supporting a range of community building activities. These activities will be centred on the specific technical goal of achieving programmable interactions between biological and artificial cells. By focusing on this specific technical challenge we hope to contribute to bridging the gap between synthetic biology from the top-down (i.e. knocking out or modifying functions of existing cells) and bottom-up synthetic biology, that is, from first principles. We believe that both approaches are important and will have a role to play in the future of synthetic biology, hence a challenge that calls for the interaction between top-down systems (modified cells) and bottom-up systems (chells, protocells) provides the ideal background against which a new research community can be built and sustained. .......................................................................................................................................

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Colyer, John Institution: University of Leeds

Oral presentation – SCN Researcher Exchange report Title: Peptide polymers assemble in response to enzyme catalysed modification Key words: peptide, phosphorylation, self assembly Abstract ....................................................................................................................................... Alpha helical coiled coil peptides were designed with a serine residue, capable of being phosphorylated, in one heptad on the hydrophobic face. Prior to phosphorylation these peptides formed coiled-coil dimers, however following phosphorylation two patterns of behaviour were seen depending on the number of heptads in the peptide. For short peptides (4.5 heptads) phosphorylation of a single (central) serine in an ‘a’ position destablised the coiled coil structure resulting in monomeric unstructured phospho-peptide sequences. This phosphorylation dependent binding and unbinding of peptides was exploited to form a FRET-based biosensor of kinase and phosphatise activity.

For peptides of longer length (5.5 heptads), phosphorylation of a single (central) serine in an ‘a’ position disrupted the former blunt-ended coiled coil dimer structure and resulted in the formation of fibres of several μm in length and variable girth. Dephosphorylation of the peptides within this fibre structure resulted in the complete dissolution of the fibre and the reappearance of soluble coiled-coil dimers. We hypothesised that coiled-coil peptides were assembling in a sticky-ended manner, in either a parallel or anti-parallel (or both) orientation. A further peptide with ‘e’ and ‘g’ position residues which strongly favour parallel orientation was also able to assemble into large fibres following phosphorylation of the single serine residue in the central heptad. Thus the control of fibre formation and dissolution could be achieved by the action of biological enzymes at physiological pH.

Yan Jin, Elinor K. Russell, Drew Thomson, Dek N. Woolfson & John Colyer

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Fletcher, Jordan Institution: School of Chemistry, University of Bristol

Oral presentation Title: Towards Synthetic Cytoskeletons Key words: Coiled coil, Self Assembly, Cytoskeleton, Peptide Vesicle Abstract ....................................................................................................................................... The growing interest in Synthetic Biology brings with it an increasing demand for robust biomolecular components. With such toolkits in hand, one could imagine combining components in a modular “plug-and-play” fashion to produce systems of increasing complexity, and to perform a variety of desired tasks. The toolkits could comprise nucleic acids, polypeptides, polysaccharides, lipids or combinations of these. As one of the best-understood protein folds, we believe the coiled coil is an ideal candidate to form the basis of such a toolkit. Coiled coils comprise two or more alpha-helices that wrap around each other to form oligomeric bundles. As such they can be considered building blocks that bring proteins together and/or act as hubs for engineering and synthetic biology applications. We are now able to reliably control, through rational sequence design, coiled-coil stability, oligomerisation, homo/heteromeric assembly, and orthogonality. In the present study we sought to use coiled coils as the basis for the development of a self-assembling synthetic cytoskeleton.

The design rationale was to combine self-assembling coiled-coil units with two- and three-fold axes of symmetry leading to a self-propagating hexagonal protein array. The first step was to design and characterise two coiled-coiled assemblies: namely, one for a stable parallel homotrimer (three-fold symmetry), and another for a parallel acidic/basic heterodimer (two-fold symmetry). Once characterised, these were linked to give two heterodimeric peptides: the homotrimer linked to the first (acidic) peptide of the heterodimer pair; and the homotrimer linked to the second (basic) peptide of the heterodimer pair. As such, we aimed to produce a regular hexagonal network with a trimeic coiled coil at the intersection of each hexagon junction and a herodimer positioned along each edge.

When these two peptides are combined in aqueous buffer, TEM shows they formed discrete spheres of 50 – 200 nm diameter with regular patterning across their surface. In experiments currently underway, we are continuing to characterise these self-assembled spheres and examining their ability to encapsulate a variety of substances.

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SCN AC, 23 – 24 September 2010 Session 3: Sci Foo or emerging topics

Sci Foo or emerging topics Open session .......................................................................................................................................

Sci Foo sessions are an informal conference format pioneered by O'Reilly Media, a leading book publisher and event organiser in the field of information technology. There is no predefined agenda; instead attendees collaboratively create one during the early stages of the conference, or at the beginning of the session. Everyone is invited to contribute, show a slide or two, or raise discuss topics that have emerged during the course of the conference. In particular early career researchers are encouraged to contribute. .......................................................................................................................................

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SCN AC, 23 – 24 September 2010 Session 4: Different perspectives

Stafford, Graham Institution: University of Sheffield

Oral presentation – Network in Synthetic Biology Title: MATEs: sticking together Key words: Tissue engineering, bacteria, bioglue, Abstract ....................................................................................................................................... The MATEs (Microbial Approaches to Tissue Engineering) network is a multidisciplinary network that has tasked itself with finding new solutions to tissue engineering problems, using synthetic biology as a platform. Our networks discussions so far have been focussed on the ultimate goal of producing a novel tissue glue that will improve adhesion of skin grafts to ‘bad’ wound beds. Any solution to this problem will of course have larger implications in the field of biomedicine and create new technologies. A number of approaches have been discussed at our two retreats. A flavour of these and the beginnings of some fledgling science will be presented.

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Kearnes, Matthew Institution: Institute of Hazard and Risk Research, University of Durham

Oral presentation Title: Upstream public engagement: Building social intelligence into new and emerging technologies Key words: public engagement, governance, policy Abstract ..................................................................................................................................... New technologies have the capacity to exacerbate existing public concerns about the relationship between science and society. Such disquiet focus on the potential for such technologies to produce novel risks and wider social and ethical concerns about the nature and pace of scientific and technological innovation. Areas of new and emerging technology have also become sites for the innovation of new techniques of scientific governance. In fields such as nanotechnology, synthetic biology and geoengineering government policy increasingly speaks of the need to incorporate public engagement, social science scholarship and ethical reflection into forms of 'responsible innovation' (Owen, 2009).

In this paper offer some reflections on the recent BBSRC/EPSRC Synthetic Biology Dialogue in the context of a broader research effort to understand and characterize emergent public responses to new technologies. I explore key findings from this research, in particular with regard to the role that public engagement plays in the new governance of science. After discussing some of the background on public engagement with new technologies, I move on to describe the fundamental dilemmas that are seen as likely to arise through scientific innovation, how regulatory and ethical oversight are seen as likely to be limiting under real life circumstances, and how this generates public doubt.

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Papachristodoulou, Antonis Institution: University of Oxford

Oral presentation – Network in Synthetic Biology Title: Robustness and Design in Synthetic Biology: RoSBNet Key words: robustness, design, control engineering, synthetic biology Abstract ....................................................................................................................................... Synthetic Biology is considered to be the “Engineering of Biology”: it aims to design new biological networks and/or redesign existing ones for new or improved functionality and performance. There are now several examples of small synthetic biology circuits that have been successfully designed and implemented in the laboratory. The underlying difficulty, however, is that most designs do not work as predicted and need to be fine-tuned on a case-by-case basis. This failure is the result of an insufficient modelling and design procedure which fails to generate synthetic biosystems which can behave in a predictable fashion. This is because most designs neglect intra- and extracellular noise, environmental uncertainty, intrinsic nonlinearities etc., and do not consider all available biological ‘knobs’ that could be used to tune the system’s performance.

RoSBNet (Robust Synthetic Biology Network) is one of the seven cross-research council funded networks in Synthetic Biology and brings together Engineers, Mathematicians, Physicists, Biochemists and researchers interested in the Ethical, Legal and Societal issues that could arise from Synthetic Biology research. The underlying challenge that we aim to address is how to develop a disciplined design process so as to ensure that synthetic biology designs work reliably and efficiently, in spite of the uncertainties of the environment they have to work in. In this talk, I will present some of the RoSBNet activities and outcomes, as well as research that we have undertaken in Oxford in that direction. .......................................................................................................................................

SCN AC, 23 – 24 September 2010 Session 5: Bimolecular components

Bath, Jonathan Institution: Oxford University

Oral presentation Title: Direct observation of the stepwise movement of a DNA motor. Key words: molecular motor, self-assembly, DNA nanotechnology Abstract .................................................................................................................................... Directed movement along a linear tracks is an essential feature of biological systems because it allows control over organization and assembly of molecular components. Construction of a synthetic molecular motor might allow us to exert similar control. DNA is a useful material for the construction of nanometre-scale machines because the strength and specificity of the interactions required for assembly and operation can be encoded in the nucleotide sequence of the component parts.

We have loaded a molecular motor at one end of a 100 nm track that is displayed along the diagonal of a rectangular scaffold. We show that the motor can move the entire length of the track taking 6 nm steps. Directed movement along the track does not require external intervention. This precise control over long-range transport is an essential component of more complex systems, for example molecular assembly lines that are programmed by instructions encoded in the nucleotide sequences of track and motor.

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Brady, Leo Institution: University of Bristol

Oral presentation Title: Protein assembly by design and semi-design Key words: Protein assembly, adhesins, coiled coil, strand swapping, X-ray crystallography. Abstract ....................................................................................................................................... Designing a protein for reversible assembly to form ordered oligomeric states requires a careful balance and understanding of non-covalent forces. Protein crystals represent a highly concentrated and semi-solid state that can unexpectedly reveal protein assembly tendencies that are not obvious from solution studies. In this talk I will present selected observations from a range of protein crystal structures that we have determined over many years. These include structures of proteins that readily undergo strand-swapping to produce metastable higher-order oligomeric forms, some of which can be stabilised through protein engineering. A recently determined structure of a trimeric coiled coil bacterial adhesin will also be described. This oligomer appears to have been fortuitously stabilised through the inclusion of a hexa-his tag, a common addition to recombinant proteins. Finally, a crystal structure will also be described of a hexameric coiled coil artificial ‘channel’. This was produced when attempting the design of lower-order forms. All of these assemblies represent reversible structures that might conceivably be incorporated into synthetic bio-structures, and include the potential to assemble and disassemble in response to common biochemical signals.

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Williams, Tom Institution: University of Sussex

Oral presentation – SCN Researcher Exchange report Title: Aβ-membrane interactions and its relation to Alzheimer’s disease Key words: Amyloid, biomimetic membranes, surface plasmon field-enhanced fluorescence, QCM, AFM, calcein leakage assay Abstract: ...................................................................................................................................... Alzheimer’s disease is characterised by the aggregation and deposition of the Aβ peptide. This 40 or 42 residue peptide is the product of the proteolysis of the Amyloid Precursor Protein membrane protein, and is able to assemble to form ordered, stable amyloid fibrils as well as small, soluble and potentially cytotoxic oligomers. The toxicity of the oligomers may be associated with the ability to bind to and affect the integrity of lipid membranes. In this novel work, we have monitored and compared the ability of the potent Aβ42 peptide and the less amyloidogenic Aβ40 peptide, to bind to dye-filled unilamellar lipid vesicles using a calcein release assay and Surface Plasmon field-enhanced fluorescence. Complementary to this work, we show, using QCM-d that the aggregation state of the peptide interacts differently to bilayers, and by AFM that oligomeric Aβ causes membrane permeation and dissolution of bilayers. We have investigated the effect of lipid vesicle composition, and binding may partly be mediated by the GM1 ganglioside receptors, whereby the interaction can be inhibited by lanthanide ions.

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Tanaka, Masayoshi Institution: University of Leeds

Oral presentation Title: Development of highly regulated, biocompatible, nanowires using tubulated liposomes Key words: Liposome, Nanowire, BAR protein Abstract ....................................................................................................................................... Nanowires display unique magnetic, catalytic, optical, and electrical characteristics that differ from the bulk material due to increased surface/volume and aspect ratios. Various nanowires have been developed with several methods such as chemical etching and vapour-liquid-solid methods. However, the utilisation of lipids (liposomes) has emerged as one of the most effective platforms for producing biocompatible nanomaterials, but the methods are currently limited to develop spherical structures and this has prevented the advantages of this synthetic method being transferred to elongated structures such as nanowires.

Within the organisms, membrane invagination processes such as endocytosis is a ubiquitous phenomena from bacteria to human. Several proteins containing Bin/Amphiphysin/Rvs (BAR) protein domains have the crucial function of membrane deformation in eukaryotic cells and this activity has also been reported for in vitro assay (deforming spherical liposome to lipid tubes). Additionally we have isolated and identified a membrane tubulation protein (MamY) from magnetotactic bacteria (used to constriction membranous organelle in which magnetite crystals are synthesized). Using the BAR and MamY proteins, we are developing a novel platform for the fabrication of nanowires within the synthesized tubulated liposomes. As a first step we have successfully produced tubulated liposomes with highly regulated width. The resulting highly uniform, tunable and biocompatible nanowires present great potential for many applications such as biological electronic devices.

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SCN AC, 23 – 24 September 2010 Poster session: PIs

Connell, Simon Institution: University of Leeds

Poster (PI) Title: Controlling the organisation and mechanics of a model cell membrane Abstract ..................................................................................................................................... In order to develop devices or analytical tools that utilise phosphollipid bilayer membranes, or to fully understand processes occurring when studying biomimetic membranes (such that have an effect on membrane integrity or organisation), it is necessary to have a knowledge of the rules governing bilayer structure. In this work I demonstrate some of those factors, such as lipid composition, temperature and sample preparation history. Detailed control of membrane organisation is demonstrated in terms of single phase fluidity, of separation into domains of controllable extent and size (from nm’s to 10’s of µm’s), and of varying phase, such as two phase liquid-solid, or liquid-liquid. Three phase domains are also demonstrated unambiguously for the first time. With this knowledge it is then possible to understand how various species, such as membrane proteins, amyloid, or even detergents, interact with membranes and affect its properties on a local or global level. This has implications in many areas, such as the link between lipid rafts and detergent resistant domains, the organisation of the bilayer in the vicinity of membrane proteins, and attack of membranes by various agents. A new method for directed domain formation is also described. Some of these ideas have recently been applied to the study of Stratum Corneum (skin) lipids, as a first step in the construction of an accurate model of the skin surface.

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Jeuken, Lars Institution: University of Leeds

Poster (PI) Title: Ubiquinol oxidase activity in native-like model-membrane systems Abstract ..................................................................................................................................... Bacteria have highly diverse and highly branched respiratory chains, which consist of a range of enzymes that transfer electrons from many different substrates into or out off a common pool of lipid soluble electron carriers, known collectively as quinones. In the aerobic respiratory pathway of Escherichia coli there are ubiquinol oxidases that catalyse the oxidation of ubiquinol to ubiquinone and reduce molecular oxygen to water. Here, two membrane-modified electrode systems are presented to study these membrane-bound and quinone converting enzymes, using a proton-pumping ubiquinol oxidase as a test system. In the first model system, planar orientated membranes are formed onto gold electrodes functionalised with cholesterol derivatives. Ubiquinol oxidase activity in these planar membranes is monitored using cyclic voltammetry. The apparent KM for oxygen was measured at 1.1±0.4 µM. Increasing the concentration of lipophilic ubiquinol (UQ) above 5-10 pmol/cm2 in the membrane leads to a decrease in cbo3 activity, indicating that the enzyme is inhibited by ubiquinol at high concentrations (substrate inhibition). In the second model-membrane system, vesicles are adsorbed intact on the electrode surface. By incorporating a pH-sensitive fluorescent dye inside the vesicles, the generation of a proton gradient (ΔpH) by ubiquinol oxidase is monitored. A ΔpH of up to 0.5 units is formed when ubiquinol oxidase is electrochemically activated.

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Papachristodoulou, Antonis Institution: University of Oxford

Poster (PI) Title: Modelling and Analysis Tools for Biochemical Networks Abstract ..................................................................................................................................... Models for biochemical networks typically take the form of coupled, high-order, nonlinear differential equations. Systems of this form can be difficult to even simulate, let alone analyze and (re)design. Furthermore, it is often the case that these models contain large numbers of parameters that are difficult to estimate from experimental data.

In this work tools for analyzing large-scale high fidelity models that are based on principles from control and dynamical systems theory are presented and applied on benchmark examples. The first technique is a structured model reduction algorithm designed to maintain the network structure of the original model. The advantage of the reduction approach taken here is that it keeps track of the worst-case error as states (species) are removed from the system. Using recently developed computational tools the reduction algorithm can be applied to nonlinear systems and error bounds calculated.

Complementary to the reduction algorithm is a framework for model decomposition. The idea behind model decomposition is to break large-scale models into a set of interacting lower-order models/subsystems. The objective of the decomposition approach is to minimize the energy transferase between these subsystems. Such an approach is beneficial as this method can be used to determine biologically meaningful subunits in an automated manner. Secondly, the analysis of the original system can be verified in a composite manner by constructing subsystem analysis certificates and integrating them at the end.

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Stainland, Sarah Institution: University of Leeds

Poster (PI) Title: Biomimetic nanomagnetic materials Abstract ....................................................................................................................................... Our group seeks to synthesis novel precise bio/mineral nanomaterial architectures for nanotechnological and biomedical applications. Inspired by nature, we are using functional proteins such as biomineralisation proteins and lipid membrane to create hybrid functional nanomaterial and arrays.

Currently our main interest is the formation of membrane surrounded magnetite nanoparticles. These are biomineralised in nature within magnetic bacteria and are termed magnetosomes. Currently we are working on a number of projects such as: using the magnetic bacterial Mms proteins to control the synthesis of enhanced and functional magnetite nanoparticles in vitro; performing these biomimetic formations on surfaces in organised and functional arrays; using these and lipid membranes to surround the particles and attach them to surfaces; Use physical and biological probes to analysis how these biomineralisation protein actually control this process so precisely.

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Turberfield, Andrew Institution: University of Oxford

Poster (PI) Title: DNA: Form & Function Abstract ....................................................................................................................................... DNA is a wonderful material for nanoscale construction: it is a structural material whose self-assembly can be programmed by making use of its information-carrying capability, and its hybridization or hydrolysis can be used as to provide energy for molecular devices. The poster will describe our recent work on self-assembled molecular structures and on molecular machinery, including systems for automated chemical synthesis and free-running molecular motors. .......................................................................................................................................

SCN AC, 23 – 24 September 2010 Poster session: PhDs and PDRAs

Baker, Emily Institution: University of Bristol

Poster (PhD and PDRA) Title: TANI-GAGAG: a self-assembling, semi-conducting peptide conjugate Abstract ....................................................................................................................................... E. G. Baker1, A. R. Thomson1, D. N. Woolfson1,2, C. F. J. Faul1

1School of Chemistry, University of Bristol, UK 2 Department of Biochemistry, University of Bristol, UK

Peptide hybrid materials are of interest in developing our understanding of self-

assembly as a route for developing new functional materials from the bottom up.

Peptides provide a framework for self-assembly governed by their secondary

structure specificity, which is achieved through using specific amino acid sequences.

Much inspiration has come from Nature where materials such as silks contain

structure-forming β-sheet motifs that possess striking mechanical properties. A five

residue peptide based on Bombyx mori silk, containing glycine and alanine repeat

units (GAGAG) was coupled to tetraaniline (TANI), a semiconducting organic

oligomer. The peptide-TANI hybrid is an attractive tecton for its self-assembling

capabilities and conductive properties. Tetraaniline can exist in three oxidation

states; acid doping of the half-oxidised emeraldine base (EB) state or oxidation of

the fully reduced leucoemeraldine base state (LEB) leads to the formation of a

conducting salt, thus the potential for a ‘smart material’ is realised where conductivity

can be tuned by adjusting the external conditions. The di-block (TANI-GAGAG) was

synthesised to high yield from peptide and TANI starting materials. IR and wide

angle x-ray (WAXS) suggest the formation of β-sheet regions within a nano-

structured material which is supported by optical and electron microscopy.

J. P. O’Brien, S. R. Fahnestock, Y. Termonia, K. H. Gardner, Adv. Mater., 1998, 10, 1185 Z. Wei, C. F. J. Faul, Macromol. Rapid Commun., 2008, 29, 280 .......................................................................................................................................

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Branson, Tom Institution: University of Leeds

Poster (PhD and PDRA) Title: Self-Assembling Virus-Like Particles Abstract ....................................................................................................................................... Nature has evolved proteins that can spontaneously self-assemble to create complex structures such as virus particles and molecular motors. These capsules and molecular machines typically have sizes of 10-100 nm and thus constitute ideal building blocks for nanotechnology. The fields of bionanoscience and synthetic biology are based on the concept that by combining biological building blocks with synthetic molecules it will be possible to construct novel nanoscale structures and machines that can do useful work. If nanoscale objects were constructed using weakly-bound ligands attached to a multivalent scaffold, it should be possible to use competing high affinity ligands to bring about disassembly, or even reorganisation of the building blocks to give new architectures.

In this project we are developing general strategies to use protein-carbohydrate interactions to construct three-dimensional nanoscale virus-like particles, which do not exist in the natural world. Ligands of various lengths, and with different carbohydrate moieties of varying affinity, have been covalently attached to the cholera toxin B-pentamer (CTB). The carbohydrate units bind into the natural binding pocket of another CTB pentamer, bringing the proteins together to form different aggregates and particles. The structures thus produced have been analysed and characterised by techniques including mass spectrometry, analytical ultracentrifugation and atomic force microscopy, as presented in the work here.

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Carew, Daniel Institution: University of Bristol

Poster (PhD and PDRA) Title: Self-Assembled Peptide Sheets from a Designed Anti-Parallel Coiled-Coil Tecton Abstract ....................................................................................................................................... Proteins have an unmatched diversity of function which, if harnessed in a regular fashion, could produce materials with new capabilities. Thus far protein folding and assembly in a directed manner to form higher order structures has proven intractable with few notable exceptions. Coiled-coil design rules for short synthetic peptides offer some insight into protein assembly and can be leveraged to understand the self-assembly rules for higher order protein based materials. Here we demonstrate that a designed anti-parallel coiled coil coupled to a central synthetic unit having a defined symmetry self-assembles to create two-dimensional peptide sheets over large areas. Such a single component system is a first step towards organizing and patterning at the nanoscale using proteins and creating complex self-assembling nanomaterials with programmed functionality.

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Nikolaos N. Daskalakis Institution: University of Leeds

Poster (PhD and PDRA) Title: Bioenergetic Processes in Model Membranes Abstract ....................................................................................................................................... Generating synthetic forms of cellular life requires performing reactions that efficiently mimic that of natural cells. Lipid vesicles’ internal aqueous compartment provides a means for encapsulation of biomolecules, chemicals and probe molecules, whereas the lipid membrane acts as a barrier to the passage of charged species. Vesicles have thus attracted a great deal of interest as nanoreactors since reactions take place in a well defined space and can be monitored in real time. Regulation of membrane transport is a challenge for synthetic biology applications. Also in living cells membrane transport is a vital process that is regulated by membrane proteins and enzymes acting as ion channels or pumps. In cell respiration, membrane-bound redox enzymes utilise the free energy generated by electron transfer reactions to translocate protons across membranes, generating a proton-motive force used in cells for ATP synthesis. Here, we have studied whether it is possible to use a surface-applied electrochemical potential to generate proton gradients in surface-adsorbed vesicles. Vesicles containing an ubiquinol oxidase, cytochrome bo3 from Escherichia coli, are adsorbed on gold electrodes, which are functionalised specifically to retain the integrity of the vesicles. These vesicles are loaded with a pH-sensitive fluorescent probe and intravesicular pH changes resulting from the activity of proton pumping enzymes are monitored in real time. The activity of the enzyme results in the formation of a proton-motive force, which can be controlled by electrochemical tuning of the lipophilic ubiquinone redox state. .......................................................................................................................................

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Edmundson, Matthew Institution: University of Cardiff

Poster (PhD and PDRA) Title: Creation of deletion and substitution libraries using TriNEx technology Abstract ....................................................................................................................................... Trinucleotide exchange (TriNEx) is a new in vitro evolution mutagenesis tool that generates novel molecular diversity in a protein of interest via the manipulation of blocks of trinuleotides. Molecular diversity is introduced using an engineered transposable element, termed MuDel, that randomly inserts into a target gene. Its removal using outside-cutting restriction endonucleases and subsequent manipulations allows the generation of protein variant libraries that sample non-standard mutations. These include amino acid deletion, domain insertion and even whole codon replacement. In the case of the latter, amber stop codons (UAG) can be incorporated in combination with orthogonal tRNA/aminoacyl-tRNA synthetase pairs to allow the evolution of proteins sampling new chemical diversity through a reprogrammed genetic code. This technology has been successfully applied to Green Fluorescent Protein (sGFP). A number of mutants with altered spectral properties were identified. The TriNEx technology behind the creation of these libraries, as well as the methods used to select in-frame mutations and incorporate unnatural amino acids, are described. .......................................................................................................................................

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Galloway, Jo Institution: University of Leeds

Poster (PhD and PDRA) Title: Arrays of Nanomagnets Formed with the Bioimineralizing protein Mms6. Abstract ....................................................................................................................................... Magnetic nanoparticles (MNPs) have many applications, such as high density data storage, targeted drug delivery and hyperthermic cancer therapy. These MNPs must have a monodisperse size and shape distribution to ensure their magnetic behaviour is consistent. To date, it has only been possible to study the bulk behaviour of MNPs. Also, this consistency often requires the use of environmentally harsh chemicals, high temperatures and elevated pressure. The biomineralizing protein Mms6 from M. magneticum AMB-1 templates the formation cubo-octahedral magnetite in vitro at ambient temperatures and pressures that are akin to those formed by the bacterium in vivo. Mms6 remains closely associated with the surface of the morphologically consistent MNPs after formation, so can be used to anchor the nanomagnets to preformed nanoscale patterns. This enables the magnetic behaviour of individual or groups of MNPs to be determined. The morphology of MNPs is determined by AFM, then the magnetic behaviour of the particles analyzed by MFM. A method that allows Mms6 to remain functional after immobilization will allow nanomagnets to form on a pre-patterned surface. This could then be used to immobilize any identified recombinant biomineralizing protein and retain its functionality to form ordered nanoscale patterns of minerals on a surface. .......................................................................................................................................

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Kendall, James Institution: University of Leeds

Poster (PhD and PDRA) Title: Electrochemical Impedance Spectroscopy As A Tool For Studying Ion Channels In Tethered Bilayer Lipid Membranes Abstract ....................................................................................................................................... Membrane proteins are the eyes and ears of all cells – they allow the cell to monitor its environment and enable communication with surrounding cells. They account for 30% of most organisms’ genomes and are key targets in pharmacology. Ion channels are of particular interest to us as they couple analyte detection with the flux of ions in the order of 10 million per second. This acts as a natural signal amplifier making these channels of particular interest in biosensing applications and allows them to be measured using electrochemical impedance spectroscopy. We are using tethered bilayer lipid membranes (tBLMs) on gold electrodes as a platform on which ion channels can be incorporated. These tBLMs are composed of two phases – a mixed self-assembled monolayer (SAM), comprised of a cholesteryl tether (EO3 cholesterol) and a hydrophilic diluent (6-mercaptohexanol); and a lipid bilayer formed via the fusion of Escherichia coli polar lipid extract vesicles. Test data has been obtained using ionophores providing us with model ion transport behaviours, and we are now in the process of incorporating several ion channels. In addition to the biosensing applications already mentioned, this work has the potential for use in high-throughput screening of pharmaceuticals. .......................................................................................................................................

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Reddington, Sam Institution: University of Cardiff

Poster (PhD and PDRA) Title: Introducing novel protein functionality via incorporation of unnatural amino acids Abstract ....................................................................................................................................... Introducing new physicochemical properties into proteins in the form of unnatural amino acids (UAA) allows the production of proteins with novel and useful properties not normally accessible in Nature. UAA incorporation represents a valuable way to study proteins and opens up new possibilities for controlling the properties of a protein through precise and defined post-translational events. This method employs an engineered, orthogonal tRNA/aminoacyl-tRNA-synthetase pair to incorporate an UAA in response to a reprogrammed codon and therefore allows UAAs to be site-specifically incorporated into proteins in vivo. We have successfully incorporated p-azidophenylalanine (azidoPhe) into positions throughout the Green Fluorescent Protein (GFP). AzidoPhe has a number of desirable properties including selective reactivity via Click chemistry and photoactivation leading to structural rearrangement and crosslinking. Incorporation of azidoPhe at the central fluorophore residue Tyr66 produced a functional protein with significantly altered spectral properties compared to wild-type GFP. Additionally, the photoactivated structural rearrangement and subsequent crosslinking of azidoPhe can be used to create GFP variants capable of switching spectral properties upon photoactivation. Introduction of azidoPhe at other positions in GFP also had a significant effect on the excitation and emission profile of the protein including a variant with red-shifted excitation. The introduction of an azido group will also allow defined attachment of desirable chemical adjuncts through Click Chemistry. This includes moieties such as polyethylene glycol (PEG) which is commonly used to improve the stability of therapeutic proteins. We show here that we can use Click Chemistry to specifically modify GFP at a defined position with a fluorescent dye molecule .......................................................................................................................................

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Towey, James Institution: University of Leeds

Poster (PhD and PDRA) Title: A Biophysical Study of the Structural Properties of the Cryoprotectant Glycerol Abstract ....................................................................................................................................... Glycerol, sometimes known as glycerin, is a polyhydric sugar alcohol that contains three hydroxyl groups and has the molecular formula C3H5(OH)3. This molecule is of significant interest given its ability to act as a protecting osmolyte: a small organic molecule that stabilises biological molecules under conditions of environmental stresses such as temperature fluctuations, pressure changes or co-solute concentration. This osmolyte is active in maintaining the structure of bio-molecules and is also thought to promote self-assembly via preferential hydration. Glycerol can also be used in the production of a synthetic analogue to DNA, known as GNA, which is used as a building block for nanofabrication techniques. Given glycerol’s role in biological and industrial processes and its potential for future applications, it is crucial to gain a complete understanding of this molecule. In particular, insight is needed into the structural properties and physical mechanisms of glycerol. We have obtained the first experimentally determined, complete structure of glycerol using neutron diffraction combined with isotope substitution. This experimental data was used to constrain a 3-dimensional computational model of the system allowing us to obtain information regarding the structure and interactions of the glycerol liquid. Contrary to theoretical predictions we find little evidence for intramolecular hydrogen bonding and around 50% more intermolecular hydrogen bonds than previously reported. Our approach demonstrates the power of producing a computational model that is consistent with experimental data. Future work will provide insight into the structural mechanisms of self-assembly within glycerol-water mixtures and the effects of temperature and concentration. .......................................................................................................................................

SCN AC, 23 – 24 September 2010 Delegate list

Delegate list Christopher Agnew University of Bristol [email protected] Emily Baker University of Bristol [email protected] George Banting University of Bristol [email protected] Jonathan Bath University of Oxford [email protected] Richard Berry University of Oxford [email protected] Aimee Boyle University of Bristol [email protected] Leo Brady University of Bristol [email protected] Tom Branson University of Leeds [email protected] Beth Bromley University of Durham [email protected] Marc Bruning University of Bristol [email protected] Daniel Carew University of Bristol [email protected] Tony Cass Imperial College London [email protected] Kevin Channon University of Cambridge [email protected] Scott Cockroft University of Edinburgh [email protected] John Colyer University of Leeds [email protected] Andrew Conn University of Bristol [email protected] Simon Connell University of Leeds [email protected] Michael Danks 4GROUND Media [email protected] Nikolaos N. Daskalakis University of Leeds [email protected] Lorna Dougan University of Leeds [email protected] Matt Edmundson University of Cardiff [email protected] Steve Evans University of Leeds [email protected] Martin Fascione University of Leeds [email protected] Jordan Fletcher University of Bristol [email protected] Jo Galloway University of Leeds [email protected] Lars Jeuken University of Leeds [email protected] yan jin University of Leeds [email protected] Ben Johnson University of Leeds [email protected] Dafydd Jones University of Cardiff [email protected] Matthew Kearnes University of Durham [email protected] James Kendall University of Leeds [email protected]

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SCN AC, 23 – 24 September 2010 Delegate list

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Natalio Krasnogor University of Nottingham [email protected] Maggie Leggett University of Bristol [email protected] David Leigh University of Edinburgh [email protected] Ewan Main Queen Mary, London [email protected] Aline Miller University of Manchester [email protected] Charlotte Millership University of Sussex [email protected] Antonis Papachristodoulou University of Oxford [email protected] JJ Phillips University of Sussex [email protected] Karen Polizzi Imperial College London [email protected] Sam Reddington University of Cardiff [email protected] James Ross University of Leeds [email protected] Jonathan Rossiter University of Bristol [email protected] David Rusling University of Southampton [email protected] Kathleen Sedgley University of Bristol [email protected] Louise Serpell University of Sussex [email protected] Thom Sharp University of Bristol [email protected] Graham Stafford University of Sheffield [email protected] Sarah Staniland University of Leeds [email protected] Michael Stockton University of Edinburgh [email protected] Masayoshi Tanaka University of Leeds [email protected] William Taylor MRC [email protected] Lai Hock TEY University of Cardiff [email protected] Andrew Thompson University of Bristol [email protected] James Towey University of Leeds [email protected] Andrew Turberfield University of Oxford [email protected] Bruce Turnbull University of Leeds [email protected] Matthew Watson University of Edinburgh M.A.Watson‐[email protected] Thomas Williams University of Sussex [email protected] Adam Wollman University of Oxford [email protected] Dek Woolfson University of Bristol [email protected]

SCN AC, 23 – 24 September 2010 Researcher exchange prize

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SCN AC, 23 – 24 September 2010 Researcher exchange prize

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If yes please provide brief details e.g., from Inaugural Meeting

Details of proposed dates of exchange

Please also provide a brief description below of the exchange project/activity (using the space provided only).

SCN AC, 23 – 24 September 2010 Feedback

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SCN AC, 23 – 24 September 2010 Feedback

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