Part:BBa_K1539021:Experience

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Applications of BBa_K1539021

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.

The primer successfully inserted the HE RBS as evidenced by gel electrophoresis and sequencing.

The PCR product was digested with EcoRI and PstI and ligated to the pSB1C3 backbone digested with the same restriction enzymes. These plasmids were used in transformations with E.coli and cultures were grown at 37°C for 16 hours then minipreped using the Qiagen Miniprep Kit in a Qiagen QiaCube. The resulting plasmids were then 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 backbone using XbaI and PstI then transformed with E.coli.

A successful transformation with a HE promoter can be visually determined by pink colonies on agar plates. When a LE promoter is inserted with the HE RBS, 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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