Team:UEA-JIC Norwich/Project

From 2011.igem.org

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Latest revision as of 21:19, 21 September 2011

University of East Anglia-JIC

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).

Retrieved from "http://2011.igem.org/Team:UEA-JIC_Norwich/Project"

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-<h1 style="font-family:verdana;color:green">Project Overview</h1>
+<h1 style="font-family:verdana;color:black">Project Overview</h1>
+<br>
+<br>
-The evolution of synthetic biology; The introduction of new photosynthetic eukaryotes as model organisms.
+<p style="color:#000000">
+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 <i>Escherichia coli</i> for use in plants.
-<p style="color:#FFFFFF">
+<p style="color:#000000">
-There are challenges using plants in iGEM, namely growth time and the complexity with adapting synthetic biology approaches for plants. However, plants are a major focus of synthetic biology due to their potential use in an array of applications from food security to synthesis of biofuels. The short time scale of the iGEM competition has often meant that plant based projects are challenging and there have been very few in previous years. 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.
-<p style="color:#FFFFFF">
-There are limitations with using plants in iGEM, namely their growth time and their transformations requiring more complex protocols. However, there are good reasons to work with plants because they have post translational modification of proteins, providing a greater range of protein synthesis. Plants are also a major focus of synthetic biology because of the interest in improving plants for crops and fuel.
 <br>
+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.
 <br>
-The aim of our project work with moss and algae is to identify a range of new Biobricks within the registry which are compatible with these species. We hope to include promoters, generators, protein coding sequences, terminators and composites within this selection. To that end, forty Biobricks were transformed into E.coli with a range of functions, from mercury detection to GFP detection proteins, and from RNA thermometers to Wintergreen scent Biobricks. We will then transform these Biobricks into both algae and moss.
+<br>
+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.
 <br>
 <br>
-We also plan to submit Biobricks containing promoters and terminators specific to both moss and algae. These will be used to attempt to increase expression of the Biobricks we’ve selected. We hope that with this information and relevant promoter and terminator Biobricks available, future teams may be able to tackle plant based iGEM projects, particularly those in moss and algae, with significant foundations already put in place as they currently are for e-coli.
+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).
 <br>
-<br>
-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 may be produced in a given bacterial species, 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.
-<br>
-<br>
-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). One other option we considered was selection by Arginine, but this would narrow down the range of possible algal species future iGEM teams could use, as a strain with a mutated Arginine Biosynthesis gene would have to be used. The Ble gene we used came from the Chlamydomonas Centre (USA). This gene has been adapted for use in eukaryotic organisms, including the insertion of two introns (see above) and the addition of a 5' and 3' untranslated region (UTR). We used PCR (polymerase chain reaction) to extract the gene from the plasmid it came in, simultaneously adding the iGEm prefix and suffix to either end. We encountered difficulties in using the Ble gene, in that it contained an illegal Xba1 site in the 3' UTR. Adhering to the Biobrick assembly standards was very important to us, as we wished to make the accessibility of these two species as easy as possible for future teams. Therefore we plan to use site directed mutagenesis to remove this site.
-<br>
-<br>
-This vector has been submitted in the form of a Biobrick. For future work, we would hope to design a new Scaffold plasmid, which would include the Bleomycin resistance cassette in place of or in addition to the antibiotic resistance coded in the iGEM plasmid. Having it placed behind the iGEM prefix and suffix would allow Biobricks to be placed directly into the plasmid without consideration for aberrant excision of the Bleomycin selection marker.
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-[[File:ALGAE_PLASMID_FOR_WIKIIII.jpg]]