Part:BBa_K1539089:Experience

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Applications of BBa_K1539089

For a proof of concept GT iGEM inserted an RBS in front of BBa_J06504, which codes for the fluorescent protein mCherry. The high efficiency RBS was inserted using this primer for PCR with New England Biolabs Q5 DNA polymerase with the following PCR settings:

98°C-30s; {98°C-10s, 60°C-20s, 72°C-20s}x31 cycles; 72°C-2min; 4°C forever.

Our RBS+mCherry plasmids were used as a template for subsequent PCR with our promoter primers.

Once a promoter primer was successfully inserted using PCR, the product was also digested and ligated to the pSB1C3 backbone using XbaI and PstI then transformed with E.coli.

A successful transformation with a HE RBS can be visually determined by very light pink colonies or normal colored colonies were seen. When an LE RBS+mCherry plasmid was used as a template in PCR, there were no discernible differences in color from a positive control colony. Successful insertion of both promoter and RBS were definitively determined by sequencing.

Flow cytometry was performed on a LE Promoter+LE RBS+mCherry culture, which yielded 91.6% of the cell population contained the mCherry protein (determined by fluorescence upon activation) with on average >17,000 fluorescent molecules per cell.

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