"
Team:DTU-Denmark-2/results/data page
From 2011.igem.org
Plug ‘n’ Play biobricks
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.
We have constructed more than 48 biobricks containing the standardized overhangs that facilitates the DNA ligase free cloning and 34 unique vectors.
The method applies long complementary overhangs on the PCR product(s) as well as the destination vector. These overhangs can anneal each other to form a stable hybridization product. The overhangs on the PCR product are custom made and independent of restriction sites, which is very convenient when working with fungi and mammalian cells.All the parts in the form of PCR-products will be distributed in microtiter plates directly ready for cloning.
The ease and fastness of Plug ’n’ Play with DNA has been demonstrated by developing a fluorescent reporter system for both Aspergillus nidulans and mammalian cells.
The Technology
The technology is based on the Uracil-Specific Excision Reagent (USER) that is an enzyme that generates a single nucleotide gap at the location of a uracil. Biobricks and vectors are constructed by designing primers for PCR that contain a uracil with additional nucleotides at the 5’ ends. and following the PCR products can directly be treated with USER™ Enzyme, resulting in unique 3´ single-stranded extensions. These unique extensions allow the correct annealing to the vector. The extensions on both biobricks and vector have been designed in such a way that the single-stranded extensions not are complementary, thereby the vector will not anneal with itself and furthermore ensures the correct orientation and placement of the inserted biobricks. The result of the USER cloning is a vector and biobricks assembled into the desired molecule, directly ready for transformation of chemically competent E. coli cells.
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 . .
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
We've also characterized the following parts
1.Main page - BBa_K678XXX
