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Plug 'n' Play Assembly Standard and BioBricks
We believe that the restriction-enzyme based assembly methods have too many drawbacks for creating advanced synthetic biology - especially in higher eukaryotes as mammals and fungi. Therefore, we have developed a novel assembly standard called Plug 'n' Play with DNA based on uracil-specific excision reagent cloning. The Plug 'n' Play assembly standard allows construction and customization of multi-part devices fast and easy.
In combination with our new assembly standard, we introduce a novel approach for the principal of BioBricks.
Our vision is that each iGEM team receives a kit containing ready to use PCR products of every single BioBrick. These BioBricks are ready for assembly by just performing a USER enzyme reaction and subsequently a traditional E. coli transformation. Hereby, the cumbersome process with restriction enzymes and ligases is avoided. This further allows time to be spent on creating innovative ideas within synthetic biology, leaving more time for characterization of BioBricks and creating innovative Genetically Engineered Machines.
The Plug 'n' Play with DNA kit contains pre-produced PCR products and back-up plasmids for each part in the kit.
So far the Plug 'n' Play with DNA kit contains 50 BioBricks and 21 plasmids for mammalian cells and Aspergilli.
How it works
PCR products are first mixed together with USER enzyme. The USER enzyme generates a single nucleotide gap at the location of the uracil in the PCR product thereby generating a 8-9 bp long complementary overhang. These overhangs can anneal each other to form a stable hybridization product. The nicks in the assembled plasmid are repaired by E. coli.
As proof of concept of our assembly standard, 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. As a proof of concept of the Plug ‘n’ Play assembly system, the Copenhagen team proved the easy assembly as well as successful function in bacteria.
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 known strong promoter. The DMKP-P6 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 known 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. We tested the suitability of the CMV promoter for the expression of enhanced GFP, YFP, CFP, and mCherry as well enhanced GFP targeted to the peroxisomes. The promoter worked well as a strong constitutive promoter.
We've also characterized the following parts
1. Main page - pJEJAM1, BBa_K678049, Plasmid for transient transfection of mammalian cells. Expression of this plasmid results in a homogeneous distribution of enhanced GFP in the cell.
2. Main page - pJEJAM2, BBa_K678050,Plasmid for transient transfection of mammalian cells. Expression of this plasmid results in the localization of enhanced GFP to the peroxisomes of the cell.
3. Main page - pJEJAM3, BBa_K678051,Plasmid for transient transfection of mammalian cells. Expression of this plasmid results in a homogeneous distribution of YFP in the cell.
4. Main page - pJEJAM4, BBa_K678052, Plasmid for transient transfection of mammalian cells. Expression of this plasmid results in a homogeneous distribution of mCherry in the cell.
5. Main page - pJEJAM5, BBa_K678053, Plasmid for transient transfection of mammalian cells. Expression of this plasmid results in a homogeneous distribution of CFP in the cell.
6. Main page - pJEJAM12, BBa_K678060, Plasmid intended for genomic integration in A. nidulans. Expression of this plasmid results in a homogeneous distribution of GFP.
7. Main page - pJEJAM13, BBa_K678061, Plasmid intended for genomic integration in A. nidulans. Expression of this plasmid results in the localization of GFP to the peroxisomes.
8. Main page - pJEJAM14, BBa_K678062, Plasmid intended for genomic integration in A. nidulans. Expression of this plasmid results in a homogeneous distribution mRFP
9. Main page - pJEJAM15, BBa_K678063, Plasmid intended for genomic integration in A. nidulans. Expression of this plasmid results in the localization of mRFP to the nucleus.
10. Main page - p68, BBa_K678070, This plasmid was used to qualitatively and quantitatively analyse the promoters DMKP-P6 and PalcA in A. nidulans
