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Team:UEA-JIC Norwich/Project
From 2011.igem.org
Project Overview
The evolution of synthetic biology; The introduction of new photosynthetic eukaryotes as model organisms.
There are challenges using plants in iGEM, namely growth time and the complexity with adapting synthetic biology approaches for plants. However, plants should be 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.
The aim of our project work with moss and algae is to produce biobricks which would aid with the incorporation of photosynthetic eukaryotes into iGEM and as such we aimed to produce promoters which could be used in both bacteria and in the selected species.
What we then planned to do was to use biobricks which give quick and definitive results, such as GFP, to allow us to test the biobricks in both the bacteria, to ensure it works and to increase the numbers of plasmid, and to then extract the plasmid from these and transform these into the eukaryotes.
One of the reasons that we decided to pursue this area of research was due to the increased versatility in eukaryotic modification of proteins. 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.
As a further part of our project, we aimed to create a plasmid which could be used as a standard in synthetic biology which had antibiotic resistance built in and the presence of the biobrick restriction sites. 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). 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.
