Team:Exeter/Lab Book

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      <a href="https://2012.igem.org/Team:Exeter/lab_book/proto"; style="color:#57b947" target="_blank">Protocols</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947" target="_blank">Single Gene Plasmids and Enzyme Characterisation</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947">Showcasing Polysaccharide Production</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947" target="_blank">Showcasing Polysaccharide Production</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947">The 3-Gene Inducible Plasmid</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947" target="_blank">The 3-Gene Inducible Plasmid</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947" target="_blank">Operon Construction</a>       
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947">Operon Construction</a>       
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947" target="_blank">Glycobase</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947">Glycobase</a>
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<p> Here we present the lab books for the following work. Please follow the links above or through the brief description below to find out more.</p><br>
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       <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a></u></b>
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       <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947" target="_blank">Showcasing Polysaccharides</a></u></b>&nbsp;|
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      &nbsp;&nbsp;&nbsp;&nbsp;|&nbsp;&nbsp;&nbsp;&nbsp;Project Lead: Alex Baldwin</p>
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      &nbsp;&nbsp;<font size="2">Contributors: Alex Clowsley, Freddie Dudbridge, Becca Philp </font></p>
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       <p> Evolution has provided us with a remarkable variety of polysaccharides that have unique properties and countless uses. Their applications are visible all around us with countless examples in medicine, industry, food, cosmetics and engineering to name a few. The aim of this mini-project was to highlight some of these important polysaccharides; Hyaluronan, Levansucrase and Cyclodextrin.</p>
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       <p>In this mini-project, I will be constructing single gene plasmids of each glycosyltransferase (GTase) with a common promoter (pBAD/araC) and terminator. Genes will be sent
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      as BioBricks to the registry immediately both with and without the pBAD/araC promoter and terminator. Single gene expression plasmids will be constructed and, time permitting,
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      measure gene expression and validate the functioning of the promoter and terminator when supplemented with L-arabinose.</p>
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      <p>Enzyme characterisation will be significant in this project in two ways. Firstly, GTases are very poorly understood and of the 128 GTases identified and known to exist in
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      the production of O-antigens of all the E.coli strains, only 20 have been characterised through published literature [1-2]. Secondly, verification of the preferred substrate
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      and terminal acceptor saccharide in addition to enzyme kinetics will be essential for the proper functioning of the dry lab database. Whilst the importance of enzyme
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      characterisation cannot be over-emphasised, it is very difficult to undertake enzyme assays because no direct change in colour of substrates/products can be detected.
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      Therefore I propose to conduct coupled enzyme assays where the production of the oligosaccharide product can be linked to another enzymatic reaction which either consumes a
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      substrate or produces a product which will lead to a colour change and a modified oligosaccharide. In addition, SDS-PAGE will be used to determine the molecular weight of each
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      GTase and compared to predicted values, as well as solubility of each GTase to check functionality in vivo.</p>
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      <p>[1] Lundborg, M., Modhuker, V. & Widmalm, G. (2010) Glycosyltransferase functions of E.coli O-antigens, Glycobiology. 20:3, pp. 366-368.</p>
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      <p>[2] Woodward, R. et al. (2010) In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz, Nature Chemical Biology. 6, pp. 418-423.</p>
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       <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947">Showcasing Polysaccharide Production</a></u></b>&nbsp;&nbsp;&nbsp;&nbsp;|
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       <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947" target="_blank">GlycoBase/GlycoWeb</a></u></b>&nbsp;|&nbsp;&nbsp;<font size="2">Contributors: Liam Stubbington, James Lynch</font></p>
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      &nbsp;&nbsp;&nbsp;&nbsp;Project Lead: Becca Philp</p>
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       <p>Synthesis of polysaccharides provides us with an endless list of applications in medicine, industry, cosmetics and engineering that we are able to demonstrate. Expression
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       <p> The purpose of the database was to improve the user friendliness of our product. In the future we envisaged a database containing thousands of enzymes, all expressible in our E.coli, leading to a wealth of sugar production possibilities.</p>
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      of Levansucrase from Bacillis subtilis into our E.coli, targeting the enzyme to outside the cell, to produce levan, a polymer of fructose. Levan is a potential glue, used by
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      Newcastle Igem 2010 (BacillaFilla) to increase the lifespan of man-made structures. Levan is also a functional biopolymer in foods, feeds, cosmetics, and the pharmaceutical
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      and chemical industries. We are separately expressing hyaluranon synthase in E coli to produce the glycosoaminoglycan, hyaluranon. Hyaluranon is an important component of the
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      human cellular matrix and has medical applications in surgery and the cosmetic industry, especially skin care. Finally we are producing cyclodextrin, a cyclic oligosaccharide
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      to exhibit the alternative structures that can be produced in E coli other than fibres. Cyclodextrins have pharmaceutical and food industry applications. These examples are
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      just a few of the diverse potential that synthetic biology can achieve.</p>
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      <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947">The 3-Gene Inducible Plasmid</a></u></b>&nbsp;&nbsp;&nbsp;&nbsp;|
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       &nbsp;&nbsp;&nbsp;&nbsp;Project Lead: Freddie Dudbridge</p>
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<p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947" target="_blank">Single Gene Plasmids and Enzyme Characterisation</a></u></b>
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       &nbsp;|&nbsp;&nbsp;<font size="2">Contributors: Freddie Dudbridge, Alex Clowsley, Alex Baldwin</font></p>
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       <p>The aim of this mini project will be to create a three gene inducible plasmid. Each gene on the plasmid will be controlled by a unique promoter, thereby giving the
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      capability to turn each gene on and off. Why is this important?: The ability to turn the genes on and off will allow the creation of a monosaccharide, a disaccharide and a
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       <p> The aim of this mini-project was the construction of single gene plasmids using the glycosyltranseferase (GTase) genes we had synthesised. The gene was to be put behind a promoter and RBS sequence, and in front of a terminator. After construction of these plasmids we wanted to determine the protein expression of the particular genes and further characterise the promoter and terminator when supplemented with appropriate inducers.
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      trisaccharide. While this in itself will not create a library of polysaccharides available from a singe E. coli, it will be a proof of concept and will sit alongside a
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      Tinkercell model showing 50 genes in a plasmid. This is important. Imagine being able to control the output of 50 genes or even 250 genes on a plasmid, each transcribing a
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      different glycosyltransferase. The result would be a small polysaccharide factory without having to create a different model every time a new bespoke polysaccharide needs to
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      be synthesised.</p>
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      <p>The project will be constructed using the Biobrick 3A assembly method. The product will be tested and then analysed by mass spectrometry to show the concept works.</p>
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       <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947">Operon Construction</a></u></b>&nbsp;&nbsp;&nbsp;&nbsp;|&nbsp;&nbsp;&nbsp;&nbsp;
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       <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947" target="_blank">Operon Construction</a></u></b>&nbsp;|&nbsp;&nbsp;
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       Project Lead: Mary Beton</p>
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       <font size="2">Contributors: Mary Beton, Freddie Dudbridge, Ryan Edginton</font></p>
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       <p>My section of the project is construction of the three glycosyltransferase operons. This is going to be done through two different assembly methods with identical aims
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       <p> The aim of this section of the project was to construct three glycosyltransferase operons. This would be done using two different assembly methods with identical aims; first by Biobrick construction and then by the Gibson assembly method. The purpose if this was for a comparison of the assembly methods intended for future use by iGEM teams. Construction of a complete operon by both methods was attempted with the hope that they yielded the predicted three polysaccharides, synthesised by our chosen sequence of glycosltransferases.</p>
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      first by biobrick construction and then by the Gibson assembly method. The purpose if this is for a comparison of the assembly methods intended for future use by iGEM teams.
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      Construction of a complete operon by either or both methods will ideally yield the predicted three polysaccharides, synthesised by the chosen sequence of glycosltransferases
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      and the wzy-dependent system. The three operon variants with similar but not identical glycosyltransferase genes demonstrate the ability to construct different monosaccharide
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      combinations within the repeat unit, an alternative approach to the three gene inducible operon project, undertaken by Freddie. The structure of the polysaccharides can be
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      analysed by mass spectrometry. If the glycosyltransferases chosen do not work as hoped and time and funding allow, there is potential to order different glycosyltransferases
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      and re-attempt the operon. Alex's work will hopefully confirm their efficacy in advance. We would hope to send the final operons as new parts to the parts registry for future
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      iGEM teams to use as a biobrick.</p>
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      <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947">Glycobase</a></u></b>&nbsp;&nbsp;&nbsp;&nbsp;|&nbsp;&nbsp;&nbsp;&nbsp;Project
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            <p><b><u><a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947" target="_blank">The 3-Gene Inducible Plasmid</a></u></b>&nbsp;|
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      Lead: Liam Stubbington</p>
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      &nbsp;&nbsp;<font size="2">Contributors: Freddie Dudbridge, Alex Clowsley, Ryan Edginton, James Lynch</font></p>
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      <p>So why do we need a database and what should it be capable of?</p>
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       <p> The aim of this mini project was to create a three gene inducible plasmid. Each gene on the plasmid would be controlled by a unique promoter, thereby giving the capability to turn each gene on and off. Why is this important? The ability to turn the genes on and off would allow the creation of a monosaccharide, a disaccharide and a trisaccharide. We hoped that this would provide a proof of concept for a larger model, maybe 100 genes in a genome with the ability to produce a polysaccharide of choice at will.</p>
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       <p>The purpose of the database is to improve the user friendliness of our product. In the future we envisage a database containing thousands of enzymes, all expressible in
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      our e-coli, leading to a wealth of sugar production possibilities.</p>
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      <p>The database serves to communicate the work done in the lab with the scientific community in the sense that the user will be able input the repeating unit of their
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      choice/design, and the database will return a list of enzymes/inducer compounds, necessary in order to produce said repeating unit.</p> 
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      <p>This will improve the efficiency of the polysaccharide lab work and speed up the process of polysaccharide production.</p> 
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      <p>It is not unforeseeable that there may be more than one enzymatic approach to producing a particular polysaccharide; as a long term aim of the database project, it may be
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      possible that our database could return some of the advantages/disadvantages of certain construction pathways.  Indeed if some repeating units are not possible, the database
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      could suggest similar or alternative products with ‘nearly’ identical properties.</p>  
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      <p>Combining this database with polysaccharide properties it could be possible to define the sugars and enzymes required based on the desired properties of the interface user.
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  <font face="Verdana" color="#57b947" size="3"><a href="https://2012.igem.org/Team:Exeter/Safety"; style="color:#57b947"><u><< Return to Safety</u></a>
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    <p><a href="https://2012.igem.org/Team:Exeter/Results"; style="color:#57b947"><u>Review Our Results >></u></a>
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    <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> &nbsp;&nbsp;|&nbsp;&nbsp;
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    <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a>  &nbsp;&nbsp;|&nbsp;&nbsp;
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    <a href="https://2012.igem.org/Team:Exeter/site_map" style="color:#57B947"><u>Site Map</u></a></p>
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Latest revision as of 02:47, 27 September 2012

ExiGEM2012 Lab Book Home

Here we present the lab books for the following work. Please follow the links above or through the brief description below to find out more.



Showcasing Polysaccharides |   Contributors: Alex Clowsley, Freddie Dudbridge, Becca Philp

Evolution has provided us with a remarkable variety of polysaccharides that have unique properties and countless uses. Their applications are visible all around us with countless examples in medicine, industry, food, cosmetics and engineering to name a few. The aim of this mini-project was to highlight some of these important polysaccharides; Hyaluronan, Levansucrase and Cyclodextrin.

GlycoBase/GlycoWeb |  Contributors: Liam Stubbington, James Lynch

The purpose of the database was to improve the user friendliness of our product. In the future we envisaged a database containing thousands of enzymes, all expressible in our E.coli, leading to a wealth of sugar production possibilities.

Single Gene Plasmids and Enzyme Characterisation  |  Contributors: Freddie Dudbridge, Alex Clowsley, Alex Baldwin

The aim of this mini-project was the construction of single gene plasmids using the glycosyltranseferase (GTase) genes we had synthesised. The gene was to be put behind a promoter and RBS sequence, and in front of a terminator. After construction of these plasmids we wanted to determine the protein expression of the particular genes and further characterise the promoter and terminator when supplemented with appropriate inducers.

Operon Construction |   Contributors: Mary Beton, Freddie Dudbridge, Ryan Edginton

The aim of this section of the project was to construct three glycosyltransferase operons. This would be done using two different assembly methods with identical aims; first by Biobrick construction and then by the Gibson assembly method. The purpose if this was for a comparison of the assembly methods intended for future use by iGEM teams. Construction of a complete operon by both methods was attempted with the hope that they yielded the predicted three polysaccharides, synthesised by our chosen sequence of glycosltransferases.

The 3-Gene Inducible Plasmid |   Contributors: Freddie Dudbridge, Alex Clowsley, Ryan Edginton, James Lynch

The aim of this mini project was to create a three gene inducible plasmid. Each gene on the plasmid would be controlled by a unique promoter, thereby giving the capability to turn each gene on and off. Why is this important? The ability to turn the genes on and off would allow the creation of a monosaccharide, a disaccharide and a trisaccharide. We hoped that this would provide a proof of concept for a larger model, maybe 100 genes in a genome with the ability to produce a polysaccharide of choice at will.
<< Return to Safety

Review Our Results >>

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