Team:UEA-JIC Norwich/Project

From 2011.igem.org

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<h1 style="font-family:verdana;color:green">Our Advisors</h1>
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<h1 style="font-family:verdana;color:green">PROJECT ABSTRACT</h1>
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<p style="color:#FFFFFF">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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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.
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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.
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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.
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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.
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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.
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<p style="color:#FFFFFF">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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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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Revision as of 08:14, 12 August 2011

University of East Anglia-JIC

UNIVERSITY OF EAST ANGLIA-JOHN INNES CENTRE


Our Advisors



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.

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.

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.

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.

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.

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. </html>

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