user group meeting - SPT Labtech...natural innovators user group meeting 21st – 22nd April 2015...
Transcript of user group meeting - SPT Labtech...natural innovators user group meeting 21st – 22nd April 2015...
natural innovators
user group meeting
21st
– 22nd
April 2015
The Talking Point Conference Centre
TTP Labtech Head Office
Melbourn Science Park, Melbourn, UK
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Programme
Tuesday 21st April
08.30 Registration and coffee
09.00 Welcome
09.15 Joerg Benz – Roche
Protein crystallisation for structure based drug design
09.45
Shilong Fan - Structural Biology Centre, Tsinghua University
The future of Chinese structure biology and why we need to set up this
platform for the Chinese biology scientist
10.15
Stephane Boivin – EMBL
Shining light with Mosquito-LCP at the EMBL crystallization facility - European
Synchrotron Radiation PETRA-III
10.45 Break
11.00
Andrew Doré – Heptares Therapeutics Ltd
Crystal Structure of a Class C GPCR – Metabotropic Glutamate receptor 5
(mGluR5)
11.30
Maria Håkansson – SARomics Biostructures
mosquito applications for crystallography and liquid dispensing for biophysics
at SARomics Biostructures
12.00 Lunch
13.00 David Hargreaves – AstraZeneca
A million mosquito tips
13.30
Marieke Lamers – Charles Rivers Laboratories
HDAC 4 crystallography to support inhibitor design as a potential therapy for
Huntington’s disease
14.00
Gebhard F.X. Schertler – Paul Scherrer Institut
Lipidic cubic phase crystallisation - from an exotic idea to a very important
method in the structural biology of membrane proteins
14.30 Break
14.45 Development team presentations and blue sky discussions
15.45 Site tour
17.00 Return coach to Cambridge
17.45 Trinity canapé reception
18.10 Cambridge & Trinity College tour
19.30 Trinity dinner
21.30 Coach back to Quy Mill and Holiday Inn hotels
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Wednesday 22nd April
08.30 Coffee
09.00 Isabel Moraes – MPL, Diamond Light Source
High-throughput membrane protein structure determination
09.30 Daniel Picot – Institut de Biologie Physico-Chemique
dragonfly and the rethinking of membrane proteins crystallisation
10.00 Break
10.15
Alexey Rak – Sanofi-Aventis
The benefits gained from using a mosquito assisted high throughput
microseeding technique
10.45 Final questions and round table discussion
12.15 Lunch
13.15 Coach back to Quy Mill and Holiday Inn hotels
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Meet the European liquid handling team:
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Joerg Benz PhD, Senior Principal Scientist, Roche Pharmaceutical Research
and Early Development, Roche Innovation Center, Basel, Switzerland
Joerg obtained his PhD in 1996 from the Max-Planck-Institute of
Biochemistry in Martinsried in the group of Robert Huber studying
structure and function of peripheral membrane proteins. After an
additional year as postdoc at the Max-Planck-Institute working on
kinases, he joined the group of Ulrich Baumann at the University of
Berne in Switzerland where he was involved in the structural
characterization of initiation events of eukaryotic translation.
In 2001, Joerg decided to leave academia and continue his career in
the pharmaceutical industry. Currently, he is heading the protein
crystallisation facility in the department of Molecular Design and Chemical Biology at Roche
Pharmaceutical Research and Early Development (pRED), Basel.
Protein crystallisation for structure based drug design
High resolution 3D X-ray structures of drug targets in the presence or absence of small
molecule ligands are a requirement for structure-based drug design. High quality crystals are
a must for the method but the production of high quality crystals is very often still a major
bottleneck in the drug-design cycle. In the presentation we will provide an overview how we
try to overcome the problem of high quality crystal generation at Roche. Major emphasis will
be given to examples for which seeding, limited in situ proteolysis and generation of several
crystal forms were important, both for soluble proteins and membrane proteins.
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Shilong Fan PhD, X-ray facility manager, Structural Biology Centre, Tsinghua
University, Beijing, China
Shilong gained his PhD in biochemistry and biophysics in 2008 from
the University of Science and Technology of China. After that, he
joined Dr. Ermanno Gherardi’s group at the Oncology department,
Cambridge University to continue structural biology research for three
years.
In 2012, Shilong returned to China and joined the Structural Biology
Centre, Tsinghua University, as X-ray facility manager.
In the last two years, Shilong has worked on estabilshing an
automated macromolecule structural biology service platform. This
includes facility service and technology service for Tsinghua University.
The future of Chinese structure biology and why we need to set up this
platform for the Chinese biology scientist
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Stephane Biovin PhD, Staff Scientist, Sample Preparation and Characterisation
Facility, European Molecular Biology Laboratory, Hamburg, Germany
Stephane Boivin obtained in 2006 his PhD from and international joint
program in chemistry from the National Scientific Research Institute
(Canada) and in biology from the University of Rouen (France). His
research projects consisted to characterise binding site of vasoactive
hormone that acts through a G-protein-coupled receptor and design
structural analogs. Following his PhD he undertook a first postdoctoral
position at the University of Texas Health Science Center at Houston
(USA).
Responsible for the crystallisation and homology modelling platform,
his main project concisted to develop and characterize catalytic antibodies (abzymes) as
vaccines to prevent or treat AIDS and Alzheimer’s disease. Stephane joined EMBL Grenoble
Oustation for his second postdoctoral position where he worked to understand the
adaptation of avian influenza A virus to the human host in a pandemic context. Since 2010,
Stephane moved to EMBL-Hamburg as staff Scientist to be in charge of the SPC facility and
high-throughput crystallisation platform.
Shining light with mosquito-LCP at the EMBL crystallisation facility - PETRA3
synchrotron
The integrated Sample Preparation and Characterisation (SPC) facility is located next to the
PETRA beamlines. It consists in a full-scale facility equipped with state-of-the-art equipment
to carry out purification and characterisation of macromolecular samples that are destined
for X-ray crystallography (MX) or small angle scattering (SAXS). In addition to the biophysics
platform, users can take advantage of our high-throughput crystallisation platform which
meet the challenges of both standard and new cutting-edge experiments for soluble and
membrane proteins, as lipid-cubic phase. Our ultimate goal is to create a pipeline from
bench to beamline. I will be presenting how mosquito-LCP play a key role in serving a mixed
community of local EMBL scientists, beamline visitors and scientists from the European
Union research area. In addition, I will present an overview of new developments for which
mosquito-LCP instrument and TTP Labtech IQ plate represent a masterpieces.
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Andy S. Doré PhD, Associate Director Crystallography, Heptares Therapeutics
Ltd., Welwyn Garden City, UK
Andy directs the crystallography group at Heptares Therapeutics
overseeing all crystallographic projects undertaken by the company.
He established the crystallography group and laboratories, including
protocols for cross platform GPCR crystallisation, runs all synchrotron
trips and his group has solved over 70 GPCR crystal structures to
date, including class A and the inaugural class B and class C receptor
structures. Prior to setting up the crystallography section at Heptares
in December 2009, he was a postdoctoral training fellow in the group
of Professor Laurence Pearl (FRS) at the Institute of Cancer Research
in London for 5 years. Andy completed his DPhil in Biochemistry and Protein Crystallography
in the laboratory of Professor Sir Tom Blundell (FRS) at the University of Cambridge in 2004
having previously worked for New England BioLabs in Boston, MA as an International
Visiting Research Scholar. His first degree specialised in Molecular Biology and Genetics.
Crystal structure of a class C GPCR – metabotropic glutamate receptor 5
(mGluR5)
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Maria Håkansson PhD, Principal Scientist, SARomics Biostructures AB, MAX IV
Laboratory Crystallisation Facility Manager, Lund, Sweden
Maria Håkansson obtained her PhD in Molecular Biophysics in 2001
from Lund University. The thesis was focused on X-ray crystallography
and was called ”Structural stuides of metal-binding proteins revealing
dynamic behaviour”. Following her PhD she took the opportunity to
learn molecular biology and protein expression as a postdoc at the
Clinical Chemistry Department at Malmö University Hospital, Sweden.
From 2006 and onwards Maria has been working for SARomics
Biostructures. She has also helped to set up the crystallisation facility
at the MAX IV Laboratory synchrotron. She has successfully worked
with crystallisation, structure determination and biophysical characterisation of many
different proteins and protein-ligand complexes.
mosquito applications for crystallography and liquid dispensing for
biophysics at SARomics Biostructures
Standard and not so standard applications of the mosquito from the daily workflow at
SARomics Biostructures will be described. Among the standard applications is crystallisation
of protein/ligand complexes ranging from small fragments to protein–protein complexes.
Seeding and additive screening belong to the common techniques employed. However we
also use the mosquito in a non-standard way as a liquid handling robot enabling easy mixing
of samples for biophysical characterisation such as dynamic light scattering and differential
scanning fluorimetry (Thermofluor).
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David Hargreaves PhD, Associate Principal Scientist, Discovery Sciences,
Structure & Biophysics, AstraZeneca, Cambridge, UK
David studied Biochemistry and Microbiology as a mature student at
the University of Sheffield after which he went on to gain a PhD in X-
ray Crystallography in Prof. David Rice’s lab. David’s thesis work
resulted in the first visualisation of the DNA Holliday Junction bound to
its partner protein RuvA. David then completed 5 years post doctoral
study on DNA helicases and plasmid maintenance systems after
which he joined AstraZeneca.
David supports small molecule drug discovery projects across
oncology working with groups in Boston and Sweden and is leading a
long running project looking at the utility of antibodies and other macromolecular entities as
tools in crystallisation.
A million mosquito tips
The mosquito has been the core piece of equipment in our High Throughput Crystallography
facilities both here in the UK, Sweden and Boston. In this talk I will describe my experiences
with it over the last 10 years which will include how we operate our standard screening
protocol and some of the more esoteric uses of the Mosquito both in crystallography and
other disciplines.
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Marieke Lamers PhD, Principle Scientist, BioFocus Discovery Services, Charles
River Laboratories, Saffron Walden, UK
Marieke joined BioFocus in September 2008 and as a group leader of
the BioFocus structural biology team has responsibility for
management of the group. In this role she is involved in the execution
and overseeing of projects concerning construct design, cloning,
expression, purification and crystallisation to enable successful protein
structure determination.
Previously Marieke was one of the founding members of Sareum Ltd, a
Structure-Based Drug Discovery Company. As Head of Protein
Sciences she was responsible for the co-ordination of the protein
expression, purification and crystallisation for both internal and external structural biology
programs. Through ten years of working in a contract research organisation, firstly with
Sareum and then with BioFocus, Marieke has interacted with a diverse customer base
including large pharmaceutical businesses and small biotech companies on a variety of
discovery research projects.
HDAC 4 crystallography to support inhibitor design as a potential therapy for
Huntington’s disease
HDAC4 crystallography at CRL was part of an integrated program in collaboration with the
CHDI Foundation, Inc. (Los Angeles, USA), to develop catalytic-site small molecule
inhibitors of the Class IIa histone deacetylases HDAC4, 5, 7 and 9. The CHDI Foundation, a
biomedical research foundation devoted to discovering Huntington’s disease-modifying
therapies, collaborated with CRL, to crystallise HDAC4 in a state amenable to drug design.
Inhibition of Class IIa HDAC enzymes have been suggested as a therapeutic strategy for a
number of diseases, including cancer, muscle wasting disorders, Huntington’s disease and
various metabolic disorders. This optimization process to develop catalytic-site small
molecule inhibitors was supported by structural biology, exemplified by the co-crystallization
of selected inhibitors with the catalytic domain of human HDAC4.
Initially the structures of wild type HDAC4 in complex with three different CHDI inhibitor
molecules were solved, but analysis of the results were complicated by the presence of
close crystal packing interactions at the active site. Subsequently, a program to develop a
more robust structural model for HDAC4 crystallography was undertaken, and this resulted
in development of a mutant enzyme crystal structure with much improved crystal packing,
allowing reliable study of enzyme ligand interaction. Through this process the mosquito
Crystal was used for primary screen set-ups and optimization studies which were crucial in
obtaining well-diffracting crystals.
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Prof Gebhard Schertler, Head of Biology and Chemistry Department, Paul
Scherrer Institut, Villigen PSI, Switzerland
Prof. Gebhard Schertler is investigating the structure and function of G
protein coupled receptors (GPCRs). When the group was located at
the MRC Laboratory for Molecular Biology (Cambridge, UK), they
focused on 3D structural analysis by X-ray and electron
crystallography and solved the atomic structures of several GPCRs
(rhodopsin, beta adrenergic receptors). With this expertise, Schertler
successfully revealed the mechanisms of light-induced rhodopsin
activation and agonist-binding to a GPCR.
Since his move to the Paul Scherrer Institute in 2009, Schertler has established an
interdisciplinary research group on GPCRs, including crystallography, electron microscopy,
NMR, biophysics and bioinformatics and a platform for protein
expression/purification/crystallization. He is also responsible for biological applications on
the Swiss Free Electron Laser (SwissFEL) and is involved in the experimental setup and
design of biology beamlines for optimization of both biomolecular nanocrystallography and
biological X-FEL imaging. Schertler is leading the Biology and Chemistry Department and is
a member of the Board of Directors at the Paul Scherrer Institute (PSI). He is also a
Professor for Structural Biology at the ETH in Zürich, Switzerland.
Lipidic cubic phase crystallisation - from an exotic idea to a very important
method in the structural biology of membrane proteins
The origin of lipidic cubic phase (LCP) crystallisation was the ingenious meeting of structural
biology with the study of lipids and lipidic substances. Rosenbusch and Landau brought
together two fields of research and created a novel crystallisation method that can fulfill at
once the optimisation of hydrophobic and hydrophilic interactions. They also were able to
develop reliable protocols to execute the experiments, and with the crystallisation of bacterial
rhodopsin with LCP they had a true breakthrough. It took many efforts from many people to
get to more automated and material saving procedures and finally first robotic automation
was possible. The mosquito LCP robot was not the first LCP robot but it was an attempt to
develop a dedicated platform optimised for the high through-put laboratory. It was based on
a prototype built by Schertler’s team at the MRC Laboratory of Molecular Biology.
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Isabel Moraes PhD, Membrane Protein Laboratory group leader, Imperial
College/Diamond Light Source, Didcot, UK
Since 2010 Isabel has led the Membrane Protein Laboratory (MPL) at
Diamond Light Source. At the MPL, she is responsible for the
management of the facility as well as its scientific research. Isabel
provides scientific advice and guidance to all projects making use of
the MPL.
She is also responsible for the establishment of internal and external
collaborations (national and international). She has established
collaborations with the Diamond MX beamline scientists regarding to
the development of new methodologies in crystallisation and structure
determination of membrane proteins.
In the last few years she has organised many courses and workshops in field of membrane
proteins in the UK and abroad. Previously to MPL, Isabel worked in many years as a
structural biologist in industry where she gained vast experience in drug discovery. Her PhD
degree and postdoctoral position were also in structural biology applied to drug discovery
projects in collaboration with industry. She enjoys working in multi-disciplinary enviroments
and being involved in scientific public events. Recently, she has been involved in the use of
X-ray Free Electron Lasers (X-FEL) in the field of membrane protein structural biology.
High-throughput membrane protein structure determination
It is estimated that nearly 30% of proteins encoded in the human genome are membrane
proteins. They perform a variety of functions including the transport of nutrients and ions,
transport of water into and out of cells, removal of waste products and toxins from cells
produce, and the use oxygen in respiration and photosynthesis. About 60% of the
commercially available drugs target membrane proteins. Therefore, the study of membrane
protein structures provides a basic understanding of life at the molecular level and helps in
the rational and targeted design of new drugs, which could reduce unwanted side effects.
The advent of the genomics and proteomics initiatives combined with high-throughput
technologies such as automation, miniaturization, integration, and third-generation
synchrotrons have enhanced membrane protein structure determination rate. Yet, many are
the obstacles a membrane protein structural biologist researcher has to face before reaching
its final destination, the 3D structure!. I will present the latest strategies regarding to the
production of suitable crystals in membrane protein structure determination alongside with
latest strategies used at synchrotron beamlines regarding to the screening and data
collection of such demanding crystals.
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Daniel Picot, CNRS Research Director, Institut de Biologie Physico-Chimique
(IBPC), Paris, France
Daniel Picot is a researcher in the Laboratory of Physical Chemistry of
Membrane Protein at the IBPC. After a PhD at the Biozentrum Basel,
he joined the group of Michael Garavito at the University of Chicago,
one of the earliest groups solely devoted to membrane protein
crystallography. He participated in early struggles there to crystallise
membrane protein and solved the structure of prostaglandin H
synthase.
In Paris, he is working on an integrated approach to study electron
transfer chains of bioenergetics membranes. He determined the
structure of the complex cytochrome b6f of oxygenic photosynthesis. His work has given him
the opportunity of tackling various crystallisation approaches and new surfactants. He
currently hopes to grasp supramolecular membrane assemblies with the help of
crystallography. At the IBPC, he has also set up and coordinated the crystallographic facility.
dragonfly and the rethinking of membrane proteins crystallisation
Crystallisation of membrane protein makes use of a large spectrum of approaches:
crystallisation in or ex micellar phases, lipidic cubic phases, sponge phases, lamellar
phases, etc. Those utilise a broad range of surfactants and cosurfactants. This nearly infinite
space search has led to a grim outlook for membrane protein crystallography that could be
only overcome by the development of very high throughput strategies. Nevertheless, it has
been recognised since the earliest day of membrane protein crystallisation that surfactants
play an important role, for example through phase transition processes. These theoretical
considerations are in agreement with early or recent survey on crystallisation condition.
Could instrumentation like the dragonfly helps to have a more rational approach? I will try to
confront earlier pre-dragonfly observations with our recently acquired instrument.
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Alexey Rak PhD, Head of Structural Biology (LGCR), Sanofi-Aventis R&D,
Vitry sur Seine, France
Dr. Alexey Rak, Ph.D.-biochemistry and biophysics, is heading the
structural biology and biophysics efforts at the LGCR (Lead Generation
to Candidate Realization) scientific platform of Sanofi R&D. He
obtained his M.Sc in biology and genetics and second M.Sc in
Biochemistry, followed by PhDs in Biochemistry and Biophysics.
He was awarded with Prize of The Association for the Advancement of
Biomedical Sciences in 2003, European Young Investigator Award in
2004 and Peter und Traudl Engelhorn Foundation research prize in
2005 for his work in the field of vesicular membrane trafficking and
small GTPases associated signaling that he conducted at Max-Planck Institute for Molecular
Physiology in Dortmund, Germany. In 2007 Dr. Rak joined Sanofi, a leading pharmaceutical
company based in Paris, France, and is now leading structural biology and biophysics based
research in small molecules and biologics therapeutic projects for various human diseases
indications at Sanofi R&D in France.
The benefits gained from using a mosquito assisted high throughput
microseeding technique
Protein crystallisation is a major bottleneck for protein structure determination, and it still
remains the least standardised part of the protein structure determination process by X-ray
crystallography. Nucleation is the first and critical step to succeed with protein crystallisation.
Crystal nuclei (seeds) can be transferred from drop to drop to increase success with the 3D
growth of crystals. This seeding technique was introduced very early and was already
mentioned in 1979 by Blundell and Johnson[1]. This method revolutionised the protein
crystallisation process.
Microseed Matrix Seeding (MMS) was introduced by C.G. Ireton and B. Stoddard [2]. Allan
D’Arcy and co-workers expanded this method by automating the procedure and seeding
directly into crystallisation screens [3]. The method has been successfully demonstrated to
be applicable for general use and has showed success on different classes of proteins in
generating new space groups, improving diffraction quality, and finding useful hits when
there were none before [4].
TTP Labtech’s mosquito instrument is found to be essential for MMS application in high
trough put industrial operations at Sanofi’s labs. mosquito’s applications for MMS
crystallisation will be discussed during the presentation.
[1]T.L. Blundell, L.N. Jonson (1976) Protein Crystallography, Academic Press, Science [2] C.G. Ireton and B. Stoddard (2004) Microseed matrix screening to improve crystals of yeast cytosine deaminase. Acta Cryst. D60, 601-605 [3] D'Arcy A, Villard F, Marsh M. (2007) An automated microseed matrix-screening method for protein crystallization. Acta Cryst. D63, 550-4. 2007 [4] D'Arcy A, Bergfors T, Cowan-Jacob SW, Marsh M.(2014) Microseed matrix screening for optimization in protein crystallization: what have we learned? Acta Crystallogr F Struct Biol Commun. 2014 Sep;70(Pt 9):1117-26
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Useful information
Attendee list
Alexey Rak Sanofi-Aventis
Aline Reynaud EPFL
Allan Surgenor Vernalis R&D
Andrew Doré Heptares Therapeutics Ltd
Annette Schousboe Petersen Novo Nordisk A/S
Artem Muravev Qvadros-Bio
Ayaka Take AsOne
Colin Levy University of Manchester
Daniel Picot Institut de Biologie Physico-Chemique
David Hargreaves AstraZeneca
Dean Derbyshire Medivir AB
Desley Pitcher AXT
Dimitri Chirgadze University of Cambridge
Dirk Gewert Horizon Discovery
Fabrice Gorrec MRC, Cambridge
Gebhard Schertler Paul Scherrer Institut
Ilka Mueller Biofocus Discovery Services - Charles Rivers Laboratories
Isabel Moraes MPL, Diamond Light Source
Joerg Benz Roche
Jorge Enrique Gonzalez Reyes Gonzalez Reyes & Cia
Julie Mosley Glaxosmithkline
Keishi Nakayama AsOne
Kor Kalk Rug
Kornelius Zeth Universidad del País Vasco/Euskal Herriko Unibertsitatea
Linda Oster AstraZeneca
Margarete Neu Glaxosmithkline
Maria Hakansson SARomics Biostructures AB
Marieke Lamers Biofocus Discovery Services - Charles Rivers Laboratories
Masahiro Matsushita AsOne
May Marsh Paul Scherrer Institut
Mindy Tan BioLab
Rafael Couňago Structural Genomics Consortium
Sarah Schulze UCB S.A. - Celltech
Sarveshwar Johri TTP Labtech India
Shilong Fan Tsinghua University
Shou Lee CSBiotech
Stephane Boivin European Molecular Biology Laboratory
Xiao Feng LBD
Ying Yang LBD
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Internet connection
Wifi name: TTP-Guest password: talkingpoint
Coach arrangements
Coaches have been arranged each day to pick up and drop off UGM guests between the
two recommended hotels (Cambridge Quy Mill hotel and Holiday Inn Lake View hotel) and
TTP Labtech.
Tuesday 21st
7.30 am pick up from Holiday Inn Lake View, 7.45 am pick up from the Cambridge
Quy Mill hotel
5pm pick up from the TalkingPoint Conference Centre to Cambridge tour and dinner
9.30/10pm return journey back to the hotels
Wednesday 22nd
7.30 am pick up from Holiday Inn Lake View, 7.45 am pick up from the Cambridge
Quy Mill hotel
1.15pm return journey back to the hotels
Parking in Cambridge
If you would like to join the Cambridge tour and Trinity College dinner but prefer to drive
in/out of Cambridge, the nearest recommended car park can be found in the Grand Arcade:
https://www.cambridge.gov.uk/car-parks-map. Please speak to the UGM coordinator for
further details on where to meet the group.
First aid assistance
First aiders can be found by dialing ‘0’ on any phone.
In the event of a fire
If you find a fire, please quickly inform a member of TTP Labtech as soon as possible, do not
attempt to put it out yourself.
In the event of a fire please make your way quickly and calmly to the fire meeting point near
the park entrance where your presence will be noted by the UGM coordinator.
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