Team:UEA-JIC Norwich/Project

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<h1 style="font-family:verdana;color:black">Project Overview</h1>
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<h1>Project Abstract.</h1>
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<p>For our project we wished to introduce two new model organisms: <i>Chlamydomonas reinhardtii</i> and <i>Physcomitrella patens</i>, an algae and a moss, respectively. Both are eukaryotic, photosynthetic organisms. At present, the majority of iGEM model organisms, and therefore the majority of the biobrick parts submitted to the registry, are prokaryotic. While these are often invaluable for a multitude of situations, such as testing protein function, they can never definitively clarify how a given gene will be expressed in a eukaryotic organism. Species specific responses to promoters, a different codon bias, or methylation can all have an adverse effect on expression, as well as a variety of other contributing factors. The use of Biobricks as an easy way of genetically manipulating organisms could one day prove to be a vital tool in the adaptation of eukaryotic species commonly used in instances such as human agriculture. The Moss and Algae we are introducing will pave the way for including plant species in the iGEM competition. We felt this would be a good direction for iGEM to take as plant genetics will always be a vital area of research for the future, impacting on areas such as crop growth, drug production and combating global warming. </p>
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<p>We plan to introduce two new destination plasmids, one for Moss, and one for Algae. These will consist of the 2011 iGEM plasmid complete with chloramphenicol resistance, and both will contain the current iGEM prefix and suffix. They will contain selection markers which can be universally used. We plan to submit a range of Biobricks within these two plasmids. These will include promoters, terminators, reporters, generators and composites. We will be testing around 60 of the current iGEM Biobricks in our two organisms and selecting those that work to be submitted. Of those that fail in our two organisms, we will attempt to either optimise them or place them behind promoters specific to each species to try and increase their expression. We also plan to introduce a series of promoters specific to our two species in these plasmids for future iGEM competitions to use. We plan to focus most on light production in the algae and moss as an example of the ability to use the Biobrick structures in these organisms. </p>
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<h2>Our Model Organisms</h2>
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Plants are an extremely versatile form of life and they are essential to the world we know.  Plants, should be a major focus for Synthetic Biology due to their potential use in an array of applications from food security to the synthesis of biofuels. However the short time scale of the iGEM competition has often meant that there have been very few in previous years.  There are also a great many challenges to using plants in iGEM including growth time and the complexity associated with adapting Synthetic Biology approaches for plants.
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As the first iGEM team at UEA and in co-operation with the JIC, we felt that we could make a significant contribution to plant based Synthetic Biology. The overall aim of our project is to help develop and where possible pioneer some of the fundamental technologies and methodologies needed to make plant based Synthetic Biology projects possible. To achieve this we hope to adapt existing Synthetic Biology approaches which are successful in <i>Escherichia coli</i> for use in plants.
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<p><h3><i>Escherichia coli.</i></h3>
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This is a single celled species of bacteria. It is a prokaryotic model organism. It is easily transformable, either by: electroporation or the use of unorthodox salts.It can also be genetically manipulated by conjugation and transduction.</p>
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<p><h3><i>Chlamydomonas reinhardtii.</i></h3>
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This is a single celled species of green algae. It is a eukaryotic, photosynthetic organism. It is easily transformable, either by: electroporation; the bacterium Agrobacterium tumorfaciens; glass beads; or by the use of a biolistic particle delivery system (gene gun).</p>
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<p><h3><i>Physcomitrella patens.</i></h3>
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To achieve the aim of our project we decided to work with <i>Physcomitrella patens</i> and <i>Chlamydomonas reinhardtii</i> with the intention of establishing these photosynthetic eukaryotes this year for future iGEM competitions. One of the reasons that we decided to pursue the introduction of our chosen photosynthetic eukaryotic species was due to the increased versatility of eukaryotic protein post-translational modifications. Eukaryotic post-translational modification can be vital for the successful expression of proteins. Post-translational methylation and glycosylation patterns are rarely conserved across species and never across the three Kingdoms of life. Thus, a eukaryotic protein can be produced in a given bacterial species by transferring the gene, but due to aberrant or non-existent methylation and glycosylation, will be unable to conduct the wild type functioning of the protein. Eukaryotic model organisms are therefore vital for reflecting the likely outcomes of intregrating systems composed of genes from an amalgamation of species when expressed in a eukaryotic organism.
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This is a multicellular species of moss. It is a eukaryotic, photosynthetic organism.</p>
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To lay the groundwork we planned to generate an array of biobricks including; promoters, terminators and other biobricks with useful properties. We carefully designed our biobricks to utilise antibiotic resistances which could be used in both bacteria and in our photosynthetic species. We needed a strategy to help us to achieve this and so we decided to use biobricks which could give quick and definitive results, such as GFP. With this approach we could test the biobricks in bacteria before trying to transform them into our plant species. At the same time we could also grow our transformed cultures and increase the amount of plasmid we had. The final step would be to transform our best biobricks into Algae and Moss for further characterisation.
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Time permitting, we also originally planned to create a plasmid with built-in antibiotic resistance and the biobrick restriction sites which could be used as a standard in Synthetic Biology. The program ApE (advanced plasmid editor) was used to visualise the plasmid. We used the Ble gene conferring the Bleomycin resistance cassette for the selection marker. This confers resistance to the Bleomycin family of antibiotics (we used phleomycin to select our transformed cells). The Ble gene we used came from the Chlamydomonas Centre (USA) and this gene has been adapted for use in eukaryotic organisms, including the insertion of two introns and the addition of a 5' and 3' untranslated region (UTR).
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Latest revision as of 21:19, 21 September 2011

University of East Anglia-JIC

UNIVERSITY OF EAST ANGLIA-JOHN INNES CENTRE

Bannerproject.jpg


Project Overview



Plants are an extremely versatile form of life and they are essential to the world we know. Plants, should be a major focus for Synthetic Biology due to their potential use in an array of applications from food security to the synthesis of biofuels. However the short time scale of the iGEM competition has often meant that there have been very few in previous years. There are also a great many challenges to using plants in iGEM including growth time and the complexity associated with adapting Synthetic Biology approaches for plants. As the first iGEM team at UEA and in co-operation with the JIC, we felt that we could make a significant contribution to plant based Synthetic Biology. The overall aim of our project is to help develop and where possible pioneer some of the fundamental technologies and methodologies needed to make plant based Synthetic Biology projects possible. To achieve this we hope to adapt existing Synthetic Biology approaches which are successful in Escherichia coli for use in plants.


To achieve the aim of our project we decided to work with Physcomitrella patens and Chlamydomonas reinhardtii with the intention of establishing these photosynthetic eukaryotes this year for future iGEM competitions. One of the reasons that we decided to pursue the introduction of our chosen photosynthetic eukaryotic species was due to the increased versatility of eukaryotic protein post-translational modifications. Eukaryotic post-translational modification can be vital for the successful expression of proteins. Post-translational methylation and glycosylation patterns are rarely conserved across species and never across the three Kingdoms of life. Thus, a eukaryotic protein can be produced in a given bacterial species by transferring the gene, but due to aberrant or non-existent methylation and glycosylation, will be unable to conduct the wild type functioning of the protein. Eukaryotic model organisms are therefore vital for reflecting the likely outcomes of intregrating systems composed of genes from an amalgamation of species when expressed in a eukaryotic organism.

To lay the groundwork we planned to generate an array of biobricks including; promoters, terminators and other biobricks with useful properties. We carefully designed our biobricks to utilise antibiotic resistances which could be used in both bacteria and in our photosynthetic species. We needed a strategy to help us to achieve this and so we decided to use biobricks which could give quick and definitive results, such as GFP. With this approach we could test the biobricks in bacteria before trying to transform them into our plant species. At the same time we could also grow our transformed cultures and increase the amount of plasmid we had. The final step would be to transform our best biobricks into Algae and Moss for further characterisation.

Time permitting, we also originally planned to create a plasmid with built-in antibiotic resistance and the biobrick restriction sites which could be used as a standard in Synthetic Biology. The program ApE (advanced plasmid editor) was used to visualise the plasmid. We used the Ble gene conferring the Bleomycin resistance cassette for the selection marker. This confers resistance to the Bleomycin family of antibiotics (we used phleomycin to select our transformed cells). The Ble gene we used came from the Chlamydomonas Centre (USA) and this gene has been adapted for use in eukaryotic organisms, including the insertion of two introns and the addition of a 5' and 3' untranslated region (UTR).

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