OpenPlant

R for Proteomics

This project brings together proteomics experts and computational biologists together to learn to use the R for Proteomics package, developed in Cambridge, integrate it into analysis pipelines to serve the proteomics community in Norwich.

The Idea

Proteomics is increasingly used in many research projects. While the throughput of mass spectrometers used in proteomics has increased in recent years, data processing work-flows are still a recognised bottleneck. Proteomics users struggle with large datasets. Slow algorithms and proprietary and free software often require manual intervention during data processing. That has a negative effect on reproducibility and throughput. Truly configurable tools to suit the changing requirements are rare.

Recently there has been a substantial development of R package 'R for Proteomics' (RfP) in Cambridge by L. Gatto et al. We believe that RfP is a powerful independent open source data pipeline that allows the development of customized work-flows. At the same time, it provides high quality visualization available in R, a facility mostly missing from other software packages. For this reason, it could complement our data processing and, if appropriate, become an alternative to our currently used software.

We would like to introduce the package, train ourselves and integrate it into our toolbox to serve the proteomics community in Norwich. The desired outcome should be increase in the reproducibility of data analyses and our ability to provide clearer results of protein identifications to the users in many projects we collaborate on. 

We have an agreement with RfP developer to come on site and provide hands on training.

The Team

Mr Jan Sklenar,
Proteomics and Mass Spectrometry Support Specialist, The Sainsbury Laboratory, Norwich

Dr Laurent Gatto,
Senior Research Associate, Department of Biochemistry, University of Cambridge

Ms Marielle Vigouroux,
Bioinformatics Support Specialist, Department of Computational and Systems Biology, John Innes Centre, Norwich

Dr Govind Chandra,
Senior Scientist, Molecular Microbiology, John Innes Centre, Norwich


Project Outputs

Project Report

This project is due to report in 2018.

Project Proposal

Original proposal and application

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Project Resources

Cell-free proteins synthesis as a resource for generating plant proteins

The purpose of this project is to set up a cell-free protein synthesis (CFPS) resource based at Norwich BioScience Institutes in collaboration with University of Cambridge.

The Idea

Cell-free protein synthesis (CFPS) has emerged as a powerful technique for on-demand, in vitro protein production which reduces labour and increases experimental throughput. However, cell-free systems can be laborious and expensive to set up and there is a shortage of publicly available data comparing different CFPS systems, particularly regarding plant proteins which can be difficult to express. This proposal will provide a resource for researchers to prototype their experimental plans without setting up the system themselves. We propose comparing an in-house generated E. coli S30 crude lysate system with a commercial wheat germ platform to quantify their ability to synthesize transcription factors and other plant proteins. This will provide data that can be used to build a simple model to predict which CFPS platform is best suited to a researcher’s needs. Additionally, two workshops (at Norwich and Cambridge) will communicate these results and provide additional expression data by crowdsourcing DNA assembly to workshop participants.

The purpose of this project is to set up a cell-free protein synthesis (CFPS) resource based at Norwich BioScience Institutes in collaboration with University of Cambridge. While there is growing interest in cell-free systems as a means for accelerating biological research, there is a higher barrier in terms of cost (£) and knowledge to setting up CFPS. Indeed, there is a shortage of publicly available information comparing the cost, yield, and flexibility of different CFPS systems. The limited data available often uses standardized non-plant reporter proteins such as green fluorescent protein (GFP) or luciferase which complicates comparison for plant biologists since plant proteins may require unique additives or an optimized folding environment for robust, soluble expression (Supplementary Figure 1 in proposal). This proposal aims to fill this knowledge gap by comparing a low-cost, in-house PANOx-SP E. coli S30 CFPS platform3 with a commercially available wheat germ4 CFPS kit from Promega.

The Team

Dr Quentin Dudley,
Postdoctoral Researcher, Engineering Biology Department, Earlham Institute, Norwich

Dr Susan Duncan,
Postdoctoral Researcher, Organisms and Ecosystems Department, Earlham Institute, Norwich

Mr Nicholas Larus-Stone,
Graduate Student, Department of Computer Science, University of Cambridge


Project Outputs

Project Report

This project is due to report in 2018.

Project Proposal

Original proposal and application

software.png

Project Resources


Banner image by Pablo Ramdohr, shared under licence CC BY 2.0 on Flickr

Comparative analysis of cell free and in planta protein synthesis systems

Our aim is to optimise a high-throughput protein synthesis method primarily for wheat transcription factors (TFs)

The Idea

This project brings together expertise from the Earlham Institute (Hall and Patron Labs), John Innes (Philippa Borrill) and the Hibberd Lab in University of Cambridge (Pallavi Singh). Our aim is to optimise a high-throughput protein synthesis method primarily for wheat transcription factors (TFs). We propose using a high throughput Golden Gate cloning strategy to create constructs that will allow us to directly compare yield from cell free and in planta protein synthesis systems. This funding would foster collaborations between groups from synergistic areas of plant biology, provide useful data for the synbio community and support future transcriptional network research in wheat.

Wheat is a primary world food crop. It’s production needs to increase to meet demand and understanding regulatory networks will be critical for designing crops for the future. Although we have good understanding of key networks in the model plant Arabidopsis, the extent to which they have been conserved in wheat remains an open question. In addition, we have very little understanding how gene regulatory networks operate across complex polypoid genomes. Phylogenetic analyses have revealed many core network transcription factor (TF) homologues, but knowledge of sequence specific binding sites and downstream targets will be required to determine the extent to which they can be considered functional orthologues. DNA Affinity Purification Sequencing (DAP-Seq) is a high throughput method that can provide both of these key pieces of information (O’Malley et al., 2016). It uses epitope tagged TFs to pull out associated DNA sequences to reveal binding sites. A recently published DAP-seq dataset for Arabidopsis transcription factors has paved the way for similar studies to be carried out in other plant species and Susan is leading a project in the Hall lab to set this up for wheat. We are requesting Open Plant funding to extend the optimisation of protein synthesis section of this technique with the aim of improving the ~30% DAP-Seq success rate reported for Arabidopsis TFs. It will allow us to extend the scope of the project and carry out a comprehensive test of three protein synthesis methods. We propose comparing cell free and in planta synthesis systems for 42 wheat TFs which include homologues of key Arabidopsis TFs involved in circadian rhythms and photosynthesis as well as a characterized TF that is known to be involved in wheat senescence.

The Team

Dr Susan Duncan,
Postdoctoral Researcher, Organisms and Ecosystems Department, Earlham Institute, Norwich

Dr Laura-Jayne Gardiner,
Postdoctoral Researcher, Organisms and Ecosystems Department, Earlham Institute, Norwich

Dr Quentin Dudley,
Postdoctoral Researcher, Engineering Biology Department, Earlham Institute, Norwich

Dr Philippa Borrill,
Research Fellow, Department of Crop Genetics, John Innes Centre

Dr Pallavi Singh,
Postdoctoral Researcher, Department of Plant Sciences, University of Cambridge


Project Outputs

Project Report

This project is due to report in 2018.

Project Proposal

Original proposal and application

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Project Resources

Actin visualization: to disclose mechanisms of host cell reorganisation during interactions with microbes

This project aims to develop a resource for actin visualization and imaging in living cells of Medicago truncatula

The Idea

The actin cytoskeleton is required for a multitude of plant cellular functions, including growth, development, cell architecture and response to microorganisms. Actin dynamics are involved in plant immunity and symbiotic interactions, to facilitate dramatic reorganization of the plant cytoskeleton. The aim of our project is to develop a multilevel resource for actin visualization and imaging in living cells of Medicago truncatula based on a genetically encoded fluorescent actin reporter. Medicago is the major model plant to study root nodule symbiosis and arbuscular mycorrhiza symbiosis as well as interactions with different leaf and root pathogens. However, actin labelling in Medicago is to date only achieved through root organ transformation. The establishment of a stable line with actin labelled in every cell including above ground tissue will be useful to a wide scientific community involved in plant-microbe interaction research as well as other research interests. As a powerful instrument it will enable new approaches and experiments, which are currently too complex for implementation. It will enable addressing new scientific questions and open problems and ultimately help to our better understanding underlying mechanisms of plant cell architecture rearrangement during interactions with leaf and root colonising microbes. Therefore, this resource will be exceptionally valuable for the development of new strategies of disease resistance breeding in crops.

The Team

Dr Aleksandr Gavrin,
Research Associate, Sainsbury Laboratory, University of Cambridge

Dr Sebastian Schornack,
Research Group Leader, Sainsbury Laboratory and Department of Plant Sciences, University of Cambridge

Prof Wendy Harwood,
Senior Scientist, Crop Transformation Group, John Innes Centre


Project Outputs

Project Report

This project is due to report in 2018.

Project Proposal

Original proposal and application

software.png

Project Resources


Banner image: Adapted from Gavrin et al., 2017. Interface Symbiotic Membrane Formation in Root Nodules of Medicago truncatula: the Role of Synaptotagmins MtSyt1, MtSyt2 and MtSyt3. Frontiers in Plant Sci.; 8:201. https://doi.org/10.3389/fpls.2017.00201

Focus Stacking for Teaching and Publication in Plant Sciences

This project aims to design a teaching tool and curriculum to teach focus stacked photography to secondary school children and undergraduates. This project follows on from Biomaker Challenge project Macrophotography of fern gametophytes using a DIY focus stacking system

The Idea

It has only recently become possible to take photographs of tiny plant specimens and have the full subject in focus. The technology is called focus stacking or deep focus photography. In this technique a large number of photographs are taken, while gradually moving the camera towards the subject. Each photograph has only some small areas in focus, and these focused areas are then cut out and merged together to make one, completely focused, image.

This technology is important for phenotyping in synthetic biology using Marchantia. It is also needed in very many other areas of biology research where the subject is very tiny. For example, in trichome research in Arabidopsis.

We aim to advance the use of deep focus photography in two ways:

  1. Develop a £100 teaching tool that can be used to teach the principles of deep focus photography, and a curriculum to accompany it.  
  2. Demonstrate and document how to convert the system so that it can take publishable photographs of the sort that can appear on the front cover of a journal, to be built for under £4000. Our system will accommodate subjects from 0.25mm to 1cm tall and beyond.

The Team

Dr Jennifer Deegan,
Visitor, Department of Plant Sciences, University of Cambridge

Dr Christopher Whitewoods,
Postdoctoral researcher, Cell and Developmental Biology, John Innes Centre

Dr Aleksandr Gavrin,
Research Associate, Sainsbury Laboratory, University of Cambridge

Mr Matthew Couchman,
Support Specialist, Computational and Systems Biology, John Innes Centre

Dr Richard Mortier,
University Lecturer, Computer Lab, University of Cambridge

Mr Tim Deegan,
External collaborator in the computing industry


Project Outputs

Project Report

This project is due to report in 2018.

Project Proposal

Original proposal and application

software.png

Project Resources

All resources are collated on this website. Documentation is also available on GITHUB, Hackster.io and youtube.


Light sheet microscopy of cell sheet folding in Volvox

Developing ligh sheet microscopy for imaging and experimentation on the freshwater alga Volvox, and the carniverous plant, Utricularia.

The Idea

We are applying for financial support by the OpenPlant Fund to help us improve our light sheet microscope. The amended setup will be used by the Cambridge group for laser ablation experiments on the freshwater alga Volvox. We are using the embryogenesis in Volvox as a model for epithelial development in metazoans. Stephanie Hoehn attended an EMBO course on modelling morphogenesis at the JIC in 2015, and is attempting to use computational modelling techniques developed by Prof. Enrico Coen's group at the JIC. She and her collaborators are combining imaging, experimental and theoretical approaches to study the so-called inversion process that bears similarity to gastrulation and neurulation. The Norwich group will use the light sheet microscope to study organs of the carnivorous plant Utricularia (Bladderwort).

The Team

Dr Stephanie Hoehn,
Postdoctoral researcher, Department of Applied Mathematics and Theoretical Physics, University of Cambridge

Dr Pierre Haas,
Postdoctoral researcher, Department of Applied Mathematics and Theoretical Physics, University of Cambridge

Mrs Karen Lee,
Research Assistant, Cell and Developmental Biology, John Innes Centre, Norwich


Project Outputs

Project Report

Summary of the project's achievements and future plans

Project Proposal

Original proposal and application

software.png

Project Resources

Details of modified LSFM setup on the OpenSPIM website, under Cambridge.


Half year report June 2017

Summary

Light sheet fluorescence microscopy (LSFM) is the state-of-the-art technique to study developmental processes in vivo. LSFM causes less photo-damage than confocal microscopy enabling longer time-lapse recordings. We had previously built a LSFM setup in the Goldstein group. The purpose of this project is to improve the quality of the generated LSFM data.

Optical sectioning is achieved by moving the sample through a light sheet and thereby creating z-stacks. In our previous setup images were recorded by a single camera. Due to light absorption and scattering the images of the sample half facing away from the camera showed a significant loss in image quality. In order to correct for this loss we have added a second camera and detection arm opposing the first one and covering the second half of the sample. This improved setup is doubling the thickness of a sample for which we can acquire useful fluorescence data. This significantly increases the variety of future applications including studies on the morphogenesis of entire embryos in the multicellular micro-alga Volvox and the development of feeding structures of the aquatic carnivorous plant Bladderwort.

Report and outcomes

Dr. Stephanie Höhn and Dr. Pierre Haas (DAMTP, Cambridge) are studying embryonic cell sheet folding events in Volvox that resemble the invagination of cell sheets in animals, e.g. during gastrulation (Fig. 1A,B) [1,2]. In collaboration with Dr. Karen Lee (John Innes Centre, Norwich) we are planning to study the development of the feeding bladders of the aquatic carnivorous plant Utricularia (Bladderwort). These bladders develop within spiral shaped structures. We are using LSFM to acquire long-term time-lapse recordings of these developmental processes (Fig. 1). Dr. Lee has visited the Goldstein lab in May 2017 to acquire preliminary data (Fig. 1C).

   
  
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  Figure 1 Light sheet fluorescence microscopy of Volvox and Utricularia (Bladderwort) with a custom-built setup. A, B:  Volvox embryo undergoing invagination. The image quality is significantly lower in the sample half facing away from the camera (B). In this project we added a second detection arm to achieve a homogeneous image quality throughout the sample. Green: Chlorophyll-autofluorescence.   C:  Utricularia spiral (dotted line) from which a feeding bladder will develop. Green: GFP-tagged cell wall marker.

Figure 1 Light sheet fluorescence microscopy of Volvox and Utricularia (Bladderwort) with a custom-built setup. A, B: Volvox embryo undergoing invagination. The image quality is significantly lower in the sample half facing away from the camera (B). In this project we added a second detection arm to achieve a homogeneous image quality throughout the sample. Green: Chlorophyll-autofluorescence.  C: Utricularia spiral (dotted line) from which a feeding bladder will develop. Green: GFP-tagged cell wall marker.

Our previous custom-built LSFM setup comprised two illumination arms and a single detection arm (Fig. 2A). A laser beam is split by a beam splitter and focused through cylindrical lenses to create two light sheets that illuminate the sample from two sides. The original detection arm is oriented perpendicularly to the light sheets. Due to light absorption and scattering the data quality for the sample half facing away from the camera was significantly lower (Fig. 1B).

In this project a second detection arm was added opposing the first one (Fig. 2B) to enable sample imaging from two sides and thereby achieving a homogeneous image quality throughout the sample. The two detection arms each consist of a detection objective, a tube that encloses the light path and functions as filter holder, a tube lens and a camera with adapter-mount. The sample chamber had to be modified to add a fourth objective (the second detection objective) and a lamp had to be designed for bright field illumination (Fig. 2C). All parts of the microscope are attached to an optical breadboard. To make space for the 2nd detection arm and camera, the setup had to be translated on the bread board. The breadboard was rotated by 90 degrees for better access. For safety reasons the laser path was changed, entailing a change in the position of the beam splitter (Fig. 2B).

   
  
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    Figure 2 Adding a second camera and detection arm to a custom-built light sheet microscope. A:  Previous LSFM setup. The laser beam is split by a beam splitter and focused through cylindrical lenses to create two light sheets that illuminate the sample from two sides. The original detection arm is oriented perpendicularly to the light sheets.  B:  The second detection arm increases the quality of 3-dimensional datasets by imaging samples from two sides. An LED lamp was designed to be temporarily inserted into the filter holder during sample positioning.

Figure 2 Adding a second camera and detection arm to a custom-built light sheet microscope. A: Previous LSFM setup. The laser beam is split by a beam splitter and focused through cylindrical lenses to create two light sheets that illuminate the sample from two sides. The original detection arm is oriented perpendicularly to the light sheets. B: The second detection arm increases the quality of 3-dimensional datasets by imaging samples from two sides. An LED lamp was designed to be temporarily inserted into the filter holder during sample positioning.

Details on our modified LSFM setup including photographs and drawings of the self-built parts have been made publicly accessible on the website of the OpenSPIM platform (see “Cambridge” on http://openspim.org/Who_has_an_OpenSPIM%3F).

Self-built parts:

  • All self-built parts were manufactured by the in-house machine shop at DAMTP.
  • Sample chamber: We designed a sample chamber to hold fluid medium with inlets for the 4 water dipping objectives.
  • Objective holder: we added a fourth objective holder to the metal case enclosing the sample chamber.
  • Brightfield lamp: The original space for brightfield illumination was lost when adding the 4th objective (Fig. 1). We explored different options to keep illumination (other than laser) during positioning of the sample:
    • Shining light through the top of the sample chamber did not work.
    • The objective holder for the 2nd detection objective was designed to allow for darkfield images.
    • We designed a flat LED lamp to fit in the filter holder, determined the required voltage and adjusted the power supply accordingly.
  • Tube to cover laser path and hold filters
  • Clamp to hold the detection arm
  • Filter cases
  • Custom size mirror posts

Purchased parts:

Prices for the second camera, camera mount adaptor, tube lens, objective and multi-wavelength emission filter were negotiated before purchasing them.

Follow on Plans:

We have previously been using 20x detection objectives with an N.A. of 0.5. In order to increase the resolution of our data for cell tracking we are planning to purchase a 40x, N.A. 0.5, water dipping objective LUMPLFLN40XW (Olympus, UK), priced £1,969.96. We are requesting the additional £1000 from the OpenPlant Fund to be allocated towards this 40x objective as soon as possible. The remaining £969.96 would be paid from internal funds.

We are currently integrating the second camera into the acquisition software. Both cameras will be controlled by the Multi-Camera adapter within MicroManager (https://micro-manager.org/).

We are very grateful that the OpenPlant Fund is enabling us to improve our LSFM setup and to expand our developmental studies in Volvox, Utricularia and other future model systems.


References

[1] Höhn S and Hallmann A. There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox. BMC Biology 9, 89 (2011).

[2] Höhn S, Honerkamp-Smith AR, Haas PA, Khuc Trong P, and Goldstein RE. Dynamics of a Volvox embryo turning itself inside out. Phys. Rev. Lett. 114, 178101 (2015).

Accessible 3D Models of Molecules

Creating 3D printed models of biological molecules for teaching and outreach.

The Idea

This project aims to create kits of 3D models of molecules for schools and outreach activities. The models will be used to facilitate the understanding of viral structures, polymers and synthetic biology projects. The kits will include complete structures and also pieces to be assembled as 3D puzzles and will be a tool for teachers and researchers to teach about their subject in an interactive manner.

The Team

Mr Roger Castells Graells,
Graduate student, Biological Chemistry, John Innes Centre, Norwich

Ms Vanessa Bueno,
Graduate student, Crop Genetics, John Innes Centre, Norwich

Ms Elisabeth Gill,
Graduate student, Department of Engineering, University of Cambridge


Project Outputs

Project Report

Summary of the project's achievements and future plans

Project Proposal

Original proposal and application

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Project Resources

For more information, see Roger's Youtube video about his work, and blog post about the success of this OpenPlant Fund project.


 

Accessible 3D Models of Molecules, the start of a global project

 

Summary

This project aims to create kits of 3D models of molecules for schools, outreach activities and scientific events. These models are used to facilitate the understanding of viral structures, polymers and synthetic biology projects. The models include structures and also pieces to be assembled as 3D puzzles and will be a tool for teachers and researchers to teach about their subject in an interactive manner.

The project has exceeded all the initial expectations. 3D printed models of viruses have been distributed among scientists and teachers from UK, Germany, Spain, Jordan and Kenya. In three months, the 3D printed models produced with this project have already reached people from three continents and the previsions are that it will continue expanding in the following months. We are receiving requests from scientists and teachers to produce more models that include, for example, viruses, proteins, nanoparticles, self-assembly models and bacteria.

Report and outcomes

With this project we have managed to set up a 3D printer and we have started printing 3D models already available in databases. Furthermore, we have generated new 3D printing models of viruses and proteins using structures of interest from databases with 3D shapes of proteins and macromolecular complexes (PDB and EMDB).

To generate the new 3D printing files we have used UCSF Chimera, Autodesk Netfabb Standard 2017, Cura and Prusa3D Slic3r.

There is available online a really useful guide: “Eduardo’s Guide for 3D Printing Proteins” (http://www.munfred.com/proteins.html), that describes how to use Chimera to generate 3D printable molecular models

To print the models we have used PLA (Polylactic acid) thermoplastic filament because it is a biodegradable and very strong material. It does not produce chemical odours during printing and there is a big range of colours available. So far, 3D printed models are made usually of a single colour. However, we plan to upgrade the printer in order to be able to print using four colours at the same time. This will open a whole range of opportunities in the design of new models since it will be possible to highlight specific parts of the models and distinguish different subunits that form the macromolecular complexes.

 Several of our 3D printed models of viruses.

Several of our 3D printed models of viruses.

The opportunity for scientists and teachers to have these 3D models is unique as it offers an invaluable tool that is having a huge impact in education and communication. The 3D models can be used in high school, where students can learn about the different shapes, sizes, structures and functions of viruses. Furthermore, they can be used to relate biological concepts with mathematics as some viruses follow mathematical patterns. For university teachers, it offers the possibility to talk in more detail about the structure of the viruses and make them more accessible. In outreach events it gives the public the chance to see, for some of them as the first time, the structure of molecules that are usually invisible and unknown for them. Viruses and proteins are in the scale of angstroms (10-10 metres) or nanometres (10-9 metres), therefore they are usually inaccessible for the general public. For scientists it is also a great tool to share their research with other scientists in more detail and in a more accessible way. These 3D printed models have already been used in outreach events, scientific meetings and international conferences.

The delivery of the 3D printer was delayed until April, therefore, the project has been developed mainly between April and June and just a fraction of the initial budget has been used. Furthermore, the process of getting familiar with the 3D printer and the programmes to generate the models have also taken quite a bit of time. Now that the 3D printer is stablished and that we have more knowledge in the use of the programmes, there is a possibility to increase the scope of the project.

Public engagement activities where the 3D printed models have been used

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 Tweets about the Pint of Science event.

Tweets about the Pint of Science event.

Pint of Science Festival - Medicinal Viruses: From Foe To Friend (16th May 2017):

Prof. George Lomonossoff and Roger Castells-Graells participated at the Pint of Science Festival in Norwich, with talks about viruses. The title of George’s talk was: “Just Eat Your Greens - A New Way of Vaccinating?”. The title of Roger’s talk was: “20,000 Leagues Under the Microscope: Viruses & Nanomachines” and the description: “Why is it important to understand the structure of viruses? How can viruses help us to build nanomachines? Can research and viruses fight diseases? Travel with us to the amazing nanoworld of viruses and discover how scientists are using them to build knowledge and new tools.”

At the event, several models of 3D printed viruses were distributed around and the public loved having the opportunity to interact with them. It was a great experience and we received really positive feedback.  Furthermore, a contest was organized by the Pint of Science team, where the public had to build a virus model using stationery materials. The winner received a 3D printed model of a virus. Overall, the attendants enjoyed and valued the opportunity to be able to play with structures of viruses and it was shared in Twitter.

3D printed virus models in Pia High School (Terrassa, Spain):

Roger presented some of the virus models in a high school with students aged 12 to 16 years old. The students enjoyed being able to handle and compare representations of real virus structures and were amazed that some of these structures were only discovered this year. When the school teacher was asked about how the use of educational 3D models in the classroom could benefit the learning process he answered that first of all it creates excitement and focuses the attention of the students. It is something completely new! It contributes to the understanding of three-dimensional models and gives the students a better sense of the reality of the object. Furthermore, it allows the students to calculate scale as it is possible to touch, measure and compare different models.