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>
+
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. Eventhough 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 struggeling 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'Play with DNA </a>assembly system.</p><br>
<font face="arial" size="3">
<font face="arial" size="3">
Line 92:
Line 92:
<li> 49 ready-to-use BioBricks.</li>
<li> 49 ready-to-use BioBricks.</li>
<li> 21 backup plasmids.</li>
<li> 21 backup plasmids.</li>
-
<li> A quick 'n' easy guide on how to customize new BioBricks or introducing specific mutations</li>
+
<li> A quick 'n' easy guide on how to customize the standard</li>
<dt>
<dt>
Line 100:
Line 100:
<p align="justify">
<p align="justify">
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.
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.
-
We have characterized the two fungal promoters, PalcA and DMKP-P6, and proved the function of the cytomegalovirus (CMV) promoter for mammalian cells.
+
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.
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.
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.
-
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.
+
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 colored U-2 SO cells.
-
Some of the submitted BioBricks have been successfully 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 <i>A. nidulans</i> and in the mammalian cell line U-2 OS without unwanted insertion and mutations.
+
Some of the submitted BioBricks have been successfully sequenced, and the data has been examinated 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.
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. Eventhough 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 struggeling with unwanted restriction sites and cumbersome assembly systems. This led us to standardize USER fusion by designing the Plug'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 colored U-2 SO cells.
Some of the submitted BioBricks have been successfully sequenced, and the data has been examinated 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.
Fullfillment 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.
"
