Recovered EGFP

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.

Naked eye examination

Single colony of Escherichia coli (BL21) transformed with plasmid containing EGFP is picked and cultured in liquid medium (2*YT). After overnight 37 degree culture, we transferred the liquid into an empty petri dish, and observed its fluorescence under a fluorescence microscope. Green fluorescence can be detected at the place of the bacteria solution while the rest of the plate as we expected (Figure 1).

PCR verification

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 (Figure 2).

Fluorescence quantification through the measurement kit

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.

Cell quantity

For OD600 measurement, we use the silica beads in 2019 iGEM measurement kit as a standard substance.

As a preparation, we have measured a particle standard curve of the silica beads from maximum concentration to 0 (pure ddH2O) and used iGEM official data processing excel to generate the particle standard curve. Then, we determined the best-fitted linear region with maximum correlation coefficient R2 (Figure 3). Before each time we measure our samples, we will first measure the OD600 of the silica beads samples whose concentration are at both ends of the best-fitted linear region, which in our case, is from 300,000,000/100μl to 18,750,000/100μl for calibration of the particle standard curve. After measuring the bacteria liquid culture samples, we will change the OD600 to the number of particles according to the calibrated standard curve.

Fluorescence

For fluorescence quantification, we use the fluorescein salt provided in 2019 iGEM measurement kit as a standard substance.

As a preparation, we have measured a fluorescence standard curve of the fluorescein salt from maximum concentration to 0 (pure PBS) and used iGEM official data processing excel to generate the fluorescence standard curve. Then, we determined the best-fitted linear region with maximum correlation coefficient R2 (Figure 4). Before each time we measure our samples, we will first measure the fluorescence intensity of the fluorescein salt samples whose concentration are at both ends of the best-fitted linear region, which in our case, is from 10 μM to 0.039 μM for calibration of the fluorescence standard curve. After measuring the bacteria liquid culture samples, we have changed the fluorescence intensity to the concentration of fluorescein salt according to the calibrated standard curve.

Normalization

Finally, we would divide the fluorescein salt concentration by the number of silica beads for our final quantified fluorescence intensity which is c[fluorescein salt]/n[silica beads] and has a unit of μM/(pcs per 100 μl).

As a preparation, we have measured a fluorescence standard curve of the fluorescein salt from maximum concentration to 0 (pure PBS) and used iGEM official data processing excel to generate the fluorescence standard curve. Then, we determined the best-fitted linear region with maximum correlation coefficient R2 (Figure 4). Before each time we measure our samples, we will first measure the fluorescence intensity of the fluorescein salt samples whose concentration are at both ends of the best-fitted linear region, which in our case, is from 10 μM to 0.039 μM for calibration of the fluorescence standard curve. After measuring the bacteria liquid culture samples, we have changed the fluorescence intensity to the concentration of fluorescein salt according to the calibrated standard curve.

SDS-PAGE

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 (Figure 5).