Part:BBa_K1539055:Experience

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Applications of BBa_K1539055

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 pink colonies on agar plates. When a LE RBS+mCherry plasmid is used as a template for the HE promoter primer during PCR, very light pink colonies or normal colored colonies were seen. Successful insertion of both promoter and RBS were definitively determined by sequencing.

Flow cytometry was performed on a HE Promoter+HE RBS+mCherry culture, which yielded 95.1% of the cell population contained the mCherry protein (determined by fluorescence upon activation) with on average >19,000 fluorescent molecules per cell.

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