Team:DTU-Denmark-2/results/data page

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Revision as of 09:31, 15 September 2011

• Home
• About us
  • The Team
  • Supervisors
  • Gallery
• The Project
  • Abstract
  • Introduction
  • The Plug 'n' Play
    • Assembly system
    • Customization
  • Other assembly systems
  • Achievements
  • Attributions
• Results
  • Background
  • Characterization
  • Proof of concept
    • Mammalian Cells
    • Fungi
  • Data page
  • Submitted Biobricks
  • Collaboration
• Safety
  • Safety
  • Safety rules
• Notebook
  • Lab notes
  • Protocols
• Our Partners
  • Sponsors
  • Advisors
  • Acknowledgements

We think that iGEM should be about combining biobricks in any thinkable way fast and easy, leaving more time for characterization of parts and focusing on the actual project. Therefore we have developed a novel assembly standard called Plug'n'Play with DNA based on uracil excision based cloning. The concept of our assembly standard is that iGEM teams receive a kit containing ready to use PCR products of every single biobrick. This allow for the teams to select the biobricks they need, mix them in a USER reaction, incubate it, and 70 min later competent E. coli cells can be transformed. The use of preproduced PCR products may be to unconventional for some people and therefore the kit also contains what we call a back-up plasmid containing all parts in the kit, so if you use up all your PCR product or you prefer to run our own PCR you simply use the back-up plasmids. The Plug'n'Play with DNA kit so far contains 49 biobricks and 21 plasmids for mammalian cells and Aspergilli.

How it works

First PCR products are mixed and treated with USER enzyme. The USER enzyme generates a single nucleotide gap at the location of the uracil in the PCR product thereby generating 8-9 bp long complementary overhangs. These overhangs can anneal each other to form a stable hybridization product. As a proof of concept we designed a reporter system for Aspergillus nidulans and mammalian cells. The figure shows an example of this where the mammalian cell line U-2 OS is transfected with a Plug'n'Play assembled plasmid expressing GFP with a targeting sequence localizing the produced GFP to the peroxisomes of the cell.

Data For Our Favorite Parts
1. Main page - DMKP-P6 promoter, BBa_K678000, The specific activity of the constitutive promoter was assayed and compared to a strong promoter. The promoter appears to be of medium strength.
2. Main page - PalcA promoter, BBa_K678001 The specific activity of the inducible promoter was assayed and compared to a strong promoter. The promoter appears to be of medium strength .
3. Main page - Device for expression of GFP in mammalian cells, BBa_K678002, This device was used to transfect the mammalian cell line U-2 OS and as expected resulted in the localization of GFP to the peroxisomes.

Data For Pre-existing parts
1. Experience - CMV promoter, BBa_J52034, This promoter was used as the regulator for our reporter system for mammalian cells and worked well. eller. We tested the suitability of the CMV promoter for the expression of enhanced GFP, YFP, CFP, mCherry and GFP targeted to the peroxisomes and it worked well.

We've also characterized the following parts
1. Main page - pJEJAM1, BBa_K678049,
2. Main page - pJEJAM2, BBa_K678050,
3. Main page - pJEJAM3, BBa_K678051,
4. Main page - pJEJAM4, BBa_K678052,
5. Main page - pJEJAM5, BBa_K678053,
6. Main page - pJEJAM12, BBa_K678060,
7. Main page - pJEJAM13, BBa_K678061,
8. Main page - pJEJAM14, BBa_K678062,
9. Main page - pJEJAM15, BBa_K678063,
10. Main page - p68, BBa_K678070,

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