Difference between revisions of "Team:Fudan-TSI/Measurement"

We focused our measurement on characterizing the fluorescence recovery of EGFP from its nonsense mutation in the following 4 ways:

1) Green fluorescence could be seen on the plate under UV light through naked eyes and recorded by a cellphone camera. Liquid culture could be placed in a culture dish and fluorescence is easily detectable under fluorescent microscopy.

2) We designed PCR primers to only amplify the recovered EGFP sequence but not the mutated version. The amplified band could be easily visualized after electrophoresis.

3) Fluorescence level was quantified through microplate reader according to fluorescein solutions and silicon beads, both standard samples are from the distributed measurement kit.

4) We ran PAGE gel of IPTG induced bacterial lysates. The mutated version produced a truncated protein at 17.8 kD, while the recovered EGFP is 26.9 kD.

We used multiple methods to ensure that EGFP is truly recovered from its nonsense mutation.

Escherichia coli (BL21) transformed with plasmid containing EGFP is coated on a petri dish and cultured for 12-14 h overnight. Then the dish lid is removed and placed on UV light. Green fluorescence is emitted from single-cell colonies and detectable through naked eyes (Fig. 1).

We designed a set of primers which cannot amplify the nonsense mutant but is able to amplify the recovered EGFP. After PCR reaction, electrophoresis is performed and the recovered EGFP band is visibly bright while the mutant band does not appear (Fig. 3).

After being sure that the fluorescence it recovered, we quantified its intensity with a microplate reader and the standard samples from distributed measurement kit. We followed the calibration protocol from measurement community.

The EGFP nonsense mutant can only express a truncated peptide of 17.8 kD, while the full-length EGFP protein is 26.9 kD, the difference between their molecular weight could be visualized through SDS-PAGE. (Fig. 5)