Team:DTU-Denmark-2/Project/achievements

From 2011.igem.org

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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.</p><br>
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The team came together during a course in mammalian cell biology in June, and that's when the idea of iGEM popped into our heads. Eventhough we started the iGEM project in 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 systems, instead of struggeling with unwanted restriction sites and limited assembly systems. This led us to the USER fusion standard assembly as a model our <a hret="https://2011.igem.org/Team:DTU-Denmark-2/Project/overview"> Plug'Play with DNA </a>assembly system.</p><br>
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<b>Our ready system consist of:</b><br>
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<b>Our Plug 'n' Play with DNA kit consist of:</b><br>
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<li> 49 BioBricks and 21 plasmids- All ready to use!.</li>  
<li> 49 BioBricks and 21 plasmids- All ready to use!.</li>  
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<li> Back-up plasmide - To ensure mutation free amplification.</li>
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<li> 21 backup plasmids.</li>
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<li> Guide on customization - All procedures only require 1 round of PCR and assembly.</li>  
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<li> A quick 'n' easy guide on how to custumize new BioBricks or introducing specific mutations</li>  
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To verify the functionality of our design, we have examined the Biobrik parts and devices, to ensure correct assembly.<br>
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We have characterized the two fungal promoters, PalcA and DMKP-P6,  and proved the function of three mammalian promoters, SV40, PGK, CMV. <br>
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To ensure that the BioBricks and devices have been assembled correctly we have tested the functionality of them in fungi and 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 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. Both promoters appeared to be of medium strengh.<br>
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We have characterized the two fungal promoters, PalcA and DMKP-P6,  and proved the function of three mammalian promoters, SV40, PGK, and CMV.  
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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, and resulted in lots of beautiful <a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian">pictures</a> of glowing peroxisomes in the U2SO cells. <br>
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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 evaluated 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 for fluorescence molecules. The transfected cells capability to express the fluorescence molecules 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 glowing peroxisomes and colored U-2 SO cells.
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All the submitted BioBricks have been sequenced, and the data have been examinated in order to verify that no mutations or incorrect insertions had occurred. The analysis of the sequencing data showed that the Plug 'n' Play parts had been correctly assembled. The sequencing and the characterization both show that the system works as expected in fungi and mammalian cells without unwanted insertion and mutations.
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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 sequencing output was examined. The results from the sequences supported the evaluation of the characterization, implying that the system works as expected in both mammalian cells and fungi without unwanted insertion and mutations.
 
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<b><h3>Medal accomplishment</h3></b>
<b><h3>Medal accomplishment</h3></b>
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<b>3. Create and share a description of the teams project using iGEM wiki and the Team's parts.</b><br>
<b>3. Create and share a description of the teams project using iGEM wiki and the Team's parts.</b><br>
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We have made a description of our project and submitted parts. can be found under Project.<br><br>
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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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<b>4. Plan to present a poster and talk at the iGem Jamboree.</b><br>
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<b>4. Plan to present a poster and talk at the iGEM Jamboree.</b><br>
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Presentation is taken form and the rehearsal for the talk at iGEM will be getting fine-tuned before the Jamboree. <br><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>
<b>5. Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Standard Biological Parts</b><br>  
<b>5. Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Standard Biological Parts</b><br>  
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We have submitted several new <a hret="https://2011.igem.org/Team:DTU-Denmark-2/results/submitted_biobricks">BioBricks</a>.  
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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>Silver</big></b><br>
<b><big>Silver</big></b><br>
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<b>1+2. Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device. Enter tis information on the"Main Page" section of the registry  </b><br>
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<b>1+2. Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device. Enter this information on the "Main Page" section of the registry  </b><br>
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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>
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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>
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<b><big>Gold</big></b><br>
<b><big>Gold</big></b><br>
<b>1:Improve an existing BioBrick Part or Device and enter this information back on the Experience Page of the Registry.</b><br>
<b>1:Improve an existing BioBrick Part or Device and enter this information back on the Experience Page of the Registry.</b><br>
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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><br>
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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. The promotor and our experience is described in <a href="http://partsregistry.org/Part:BBa_J52034:Experience"> Experience</a>. <br><br>
<b>2: Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system. </b><br>
<b>2: Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system. </b><br>
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We have helped iGEM 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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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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<b>3: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>
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Revision as of 15:27, 18 September 2011



Achievements


The team came together during a course in mammalian cell biology in June, and that's when the idea of iGEM popped into our heads. Eventhough we started the iGEM project in 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 systems, instead of struggeling with unwanted restriction sites and limited assembly systems. This led us to the USER fusion standard assembly as a model our Plug'Play with DNA assembly system.


Our Plug 'n' Play with DNA kit consist of:
  • 49 BioBricks and 21 plasmids- All ready to use!.
  • 21 backup plasmids.
  • A quick 'n' easy guide on how to custumize new BioBricks or introducing specific mutations

    • To ensure that the BioBricks and devices have been assembled correctly we have tested the functionality of them in fungi and in mammalian cells. We have characterized the two fungal promoters, PalcA and DMKP-P6, and proved the function of three mammalian promoters, SV40, PGK, and CMV. The characterization of the fungal promoters was successfully evaluated 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 for fluorescence molecules. The transfected cells capability to express the fluorescence molecules was investigated with confocal microscopy, which resulted in amazing pictures of glowing peroxisomes and colored U-2 SO cells. All the submitted BioBricks have been sequenced, and the data have been examinated in order to verify that no mutations or incorrect insertions had occurred. The analysis of the sequencing data showed that the Plug 'n' Play parts had been correctly assembled. The sequencing and the characterization both show that the system works as expected in fungi and mammalian cells without unwanted insertion and mutations.



    Medal accomplishment


    Bronze
    1. Team Registration
    All team members are registrated

    2. Complete judging form
    Judgingform will be completed as soon as possible.

    3. Create and share a description of the teams project using iGEM wiki and the Team's parts.
    We have made a description of our project (see The project), and submitted parts can be found under Results.

    4. Plan to present a poster and 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.

    5. Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Standard Biological Parts
    We have submitted 49 new BioBricks.


    Silver
    1+2. Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device. Enter this information on the "Main Page" section of the registry
    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


    Gold
    1:Improve an existing BioBrick Part or Device and enter this information back on the Experience Page of the Registry.
    We used the CMV promoter, BBa_J52034 , as the regulator for our fluorescence reporters, and it worked well. The promotor and our experience is described in Experience.

    2: 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.

    Retrieved from "http://2011.igem.org/Team:DTU-Denmark-2/Project/achievements"