<a href="https://2011.igem.org/Team:DTU-Denmark-2/Project/achievements#Fulfilment of medal requirements" class="h1"> Fulfilment of medal requirements</a><br><br>
The team came together during a course in mammalian cell biology in June, and that was when the idea of iGEM popped into our heads. Even though we started the iGEM project late June, we have managed to design a fully standardized cloning system called Plug'n Play with DNA. We thought that time should be spent on making cool iGEM projects, instead of struggling with unwanted restriction sites and cumbersome assembly systems. This led us to standardize USER fusion by designing the<a hret="https://2011.igem.org/Team:DTU-Denmark-2/Project/overview"> Plug'n'Play with DNA </a>assembly system.</p><br>
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Even though we first started the iGEM project in the end of June, we have managed to design a fully standardized cloning system called Plug'n Play with DNA. We think that iGEM should be about fast and easy assembling of BioBricks, which led us to use features from the USER fusion standard assembly to model our <a hret="https://2011.igem.org/Team:DTU-Denmark-2/Project/overview"> Plug'Play with DNA </a>assembly system. Our ready system consist of:
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<li> 49 BioBricks and 21 plasmids- All ready to use!.
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<b>Our Plug 'n' Play with DNA kit consist of:</b><br>
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<li> Back-up plasmide - To ensure mutation free amplification.
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<li> Guide on customization - All procedures only require 1 round of PCR and assembly
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<li> 49 ready-to-use BioBricks.</li>
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<li> 21 backup plasmids.</li>
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<li> A quick 'n' easy guide on how to customize the standard</li>
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To ensure that the BioBricks and devices have been assembled correctly we have tested the functionality of them in <i>Aspergillus nidulans</i> and in mammalian cells.
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We have characterized the two fungal promoters, P<i>alcA</i> and DMKP-P6, and demonstrated function of the cytomegalovirus (CMV) promoter in mammalian cells.
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The <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Characterisation">characterization</a> of the fungal promoters was successfully performed with an X-gal analysis, where the expression of ß-galactosidase results in blue colonies. Furthermore, quantitative ß-galactosidase and Bradford assays were performed in order to measure protein production and ß-galactosidase activity. Both promoters proved to be of medium strength.
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For demonstration of the Plug' Play with DNA system in <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian">mammalian cell</a> lines, U-2 OS was transfected with plasmids containing genes encoding fluorescent proteins. The transfected cells capability to express the fluorescent proteins was investigated with confocal microscopy, which resulted in amazing <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian">pictures</a> of fluorescent peroxisomes and coloured U-2 SO cells.
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Some of the submitted BioBricks have been successfully sequenced, and the data has been examined in order to verify that no mutations or incorrect insertions had occurred. The analysis of the sequencing data showed that all the sequenced Plug 'n' Play BioBricks had been correctly assembled without mutations. The sequencing and the characterization both showed that the system works as expected in <i>A. nidulans</i> and in the mammalian cell line U-2 OS.
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To verify the function of our design we examined various Biobriks, parts and devices, by experimental procedures described under protocols. .<br>
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We have characterized the two fungal promoters, PalcA and DMKP-P6, and proved function of three mammalian promoters, SV40, PGK, CMV. <br>
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<a name="Fulfilment of medal requirements"></a><h1><b>Fulfilment of medal requirements</b></h1>
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The <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Characterisation">characterization</a> of the fungal promoters was qualitative evaluated with an X-gal analysis, where the expression of ß-galactosidase results in blue colonies. Furthermore, quantitative ß-galactosidase and Bradford assay was performed to measure the protein production and ß-galactosidase activity.<br>
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To demonstrate Plug' Play with DNA also was applicable in <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian">mammalian cell</a> lines, the transfected cells capability to express the fluorescence molecules was investigated with confocal microscopy.<br>
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To ensure that there had been no occurrence of mutation and incorrect insertions in the BioBricks, they was sequenced and the resulting result was examined. The results from the sequencing supported the evaluation and characterization, implying that the system works as expected in both mammalian cells and fungi
We have made a description of our project (see The project), and submitted parts can be found under Results.<br><br>
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<input type="checkbox" checked disabled="true"><b> Present a poster and a talk at the iGEM Jamboree</b><br>
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We plan to make an excellent presentation and a magnificent poster that will be transported on first class from Denmark to Amsterdam right before the Jamboree. <br><br>
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<input type="checkbox" checked disabled="true"> <b>At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.</b><br>
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We have submitted 49 new <a hret="https://2011.igem.org/Team:DTU-Denmark-2/results/submitted_biobricks">BioBricks</a>.
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<b><big>Medal accomplishment</big></b>
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<b>Silver</b><br> <br>
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<input type="checkbox" checked disabled="true"><b>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected</b><br>
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We have demonstrated the function of the device, <a hret=!http://partsregistry.org/Part:BBa_K678002"> BBa_K678002</a>, that has been transfected into the mammalian cell line, U-2 OS. Furthermore, characterization of fungal promoter parts have been executed. These results can be viewed at our homepage or in the BioBrick registry for <a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a> and <a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a><br><br>
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<input type="checkbox" checked disabled="true"><b>Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.</b><br>
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<b>Bronze</b><br>
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1-3: Team registration, judging form and team wiki was completed in time.<br>
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4: Presentation is taken form and the rehearsal for the talk at iGEM is getting fine-tuned. <br>
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5: We used the CMV promoter, <a href="http://partsregistry.org/Part:BBa_J52034"> BBa_J52034 </a>, as the regulator for our fluorescence reporters, and it worked well. Announced under <a href="http://partsregistry.org/Part:BBa_J52034:Experience"> Experience</a> <br>
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<b>Silver</b><br>
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1-2: We have, among other, demonstrated the function of the device, <a hret=!http://partsregistry.org/Part:BBa_K678002!> BBa_K678002</a>, transfected into mammalian cell lines. Furthermore, characterization of fungal promoter parts has been executed, and the results can be viewed at our homepage or in the BioBrick registry for <a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a> and <a href="http://partsregistry.org/Part:BBa_K678001>BBa_K678001</a><br>
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<b>Gold</b>
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2: We have helped iGEM copenhagen with the design of primers for heir BioBricks.<br>
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<b>Gold</b> <br>
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<input type="checkbox" checked disabled="true"><b>Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year) and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry), and don't forget to create a new registry page for the improved part.</b><br><br>
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<input type="checkbox" checked disabled="true"><b>Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.</b><br>We have helped the iGEM team Copenhagen with the design and assembly of some of their <a href="https://2011.igem.org/Team:Copenhagen/Project/Data">BioBricks</a>.<br><br>
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<input type="checkbox" disabled="true"><b>Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.</b><br><br>
The team came together during a course in mammalian cell biology in June, and that was when the idea of iGEM popped into our heads. Even though we started the iGEM project late June, we have managed to design a fully standardized cloning system called Plug'n Play with DNA. We thought that time should be spent on making cool iGEM projects, instead of struggling with unwanted restriction sites and cumbersome assembly systems. This led us to standardize USER fusion by designing the Plug'n'Play with DNA assembly system.
Our Plug 'n' Play with DNA kit consist of:
49 ready-to-use BioBricks.
21 backup plasmids.
A quick 'n' easy guide on how to customize the standard
To ensure that the BioBricks and devices have been assembled correctly we have tested the functionality of them in Aspergillus nidulans and in mammalian cells.
We have characterized the two fungal promoters, PalcA and DMKP-P6, and demonstrated function of the cytomegalovirus (CMV) promoter in mammalian cells.
The characterization of the fungal promoters was successfully performed with an X-gal analysis, where the expression of ß-galactosidase results in blue colonies. Furthermore, quantitative ß-galactosidase and Bradford assays were performed in order to measure protein production and ß-galactosidase activity. Both promoters proved to be of medium strength.
For demonstration of the Plug' Play with DNA system in mammalian cell lines, U-2 OS was transfected with plasmids containing genes encoding fluorescent proteins. The transfected cells capability to express the fluorescent proteins was investigated with confocal microscopy, which resulted in amazing pictures of fluorescent peroxisomes and coloured U-2 SO cells.
Some of the submitted BioBricks have been successfully sequenced, and the data has been examined in order to verify that no mutations or incorrect insertions had occurred. The analysis of the sequencing data showed that all the sequenced Plug 'n' Play BioBricks had been correctly assembled without mutations. The sequencing and the characterization both showed that the system works as expected in A. nidulans and in the mammalian cell line U-2 OS.
Fulfilment of medal requirements
Bronze
Team registration
Complete Judging form
Team Wiki
We have made a description of our project (see The project), and submitted parts can be found under Results.
Present a poster and a talk at the iGEM Jamboree
We plan to make an excellent presentation and a magnificent poster that will be transported on first class from Denmark to Amsterdam right before the Jamboree.
At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.
We have submitted 49 new BioBricks.
Silver
Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected
We have demonstrated the function of the device, BBa_K678002, that has been transfected into the mammalian cell line, U-2 OS. Furthermore, characterization of fungal promoter parts have been executed. These results can be viewed at our homepage or in the BioBrick registry for BBa_K678000 and BBa_K678001
Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.
Gold
Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year) and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry), and don't forget to create a new registry page for the improved part.
Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system. We have helped the iGEM team Copenhagen with the design and assembly of some of their BioBricks.
Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.
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