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Plug 'n' Play BioBricks and Assembly Standard
We believe that the conventional restriction-enzyme based assembly method, which is standard BioBrick assembly, have too many drawbacks for creating synthetic biology. This is specially the case with synthetic biology in higher eukaryotes as mammals and fungi.
We have as a result, developed a novel assembly standard called Plug ‘n’ Play with DNA based on uracil excision based cloning. The Plug ‘n’ Play assembly standard allows rapid construction of multi-part devices fast and easy.
In combination with our new assembly standard, we further introduce a novel approach for the comprehension principal of BioBricks.
Our vision for the BioBricks, is that each iGEM team receive 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 restrictions enzymes and ligases is avoided. This further allows time to be used on creating innovative ideas within synthetic biology, leaving more time for actual characterization of BioBricks and focusing on the fun of making Genetically Engineered Machines.
The Plug ‘n’ Play with DNA kit contains pre-produced PCR products, which may be to unconventional for some people and therefore our kit also contains, what we call a back-up plasmid for each part in the kit.
The Plug ‘n’ Play with DNA kit so far 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 repair by the E. coli organisme
As a proof of concept of our assembly standard and the Plug ‘n’ Play kit, 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 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
