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Team:Valencia UPV/Project/interlab
From 2014.igem.org
Interlab Study
Our team decided to participate in the iGEM 2014 Measurement Interlab Study, consisting on obtaining fluorescence data for three genetic devices expressing GFP. Those devices were built following BioBricks assembly protocols and checked via restriction map. All the protocols can be consulted in the appropiate section of our notebook:
https://2014.igem.org/Team:Valencia_UPV/Notebook_wetlab.html#Measurement_Interlab_Study.
Figure 1. Restriction map of devices BBa_I20260, BBa_J23101+BBa_E0240 and BBa_J23115+BBa_E0240. Expected bands for BBa_J23101+BBa_E0240 and BBa_J23115+BBa_E0240 are 2046 and 943 with NotI and 1991 and 998 with NcoI. Expected bands for BBa_I20620 are 2726 and 943 with NotI and 3296, 373 with NcoI. Markers correspond to GeneRuler 100 bp DNA Ladder (100 bp) and GeneRuler 1 kb DNA Ladder, (1 kb) from Thermo Scientific.
In our lab, fluorescence and absorbance of three technical samples for each device and a negative control consisting on untransformed DH5-alpha cells were measured. Fluorescence data was obtained using a GloMax-Multi Detection System from Promega fluorometer with the Blue optical kit configured to measure each of the samples three times and display an average of those values. A Biowave CO 8000 spectrophotometer from Biochrom was used to measure absorbance at 600 nm, which is proportional to cell concentration. The ratio between fluorescence an optical density was calculated as a way of normalizing the data obtained.
Table 1. Measurements of fluorescence and optical density for each sample.
| Sample | Fluorescence | Optical density | Fluorescence / Optical density | |
|---|---|---|---|---|
| Negative control | (1) | 1.085 | 0.38 | 2.854 |
| (2) | 1.036 | 0.35 | 2.959 | |
| (3) | 1.076 | 0.39 | 2.759 | |
| BBa_I20260 | (1) | 4.907 | 0.36 | 13.632 |
| (2) | 4.754 | 0.34 | 13.981 | |
| (3) | 3.494 | 0.26 | 13.439 | |
| BBa_J23101 + BBa_E0240 | (1) | 57.393 | 0.43 | 133.471 |
| (2) | 61.622 | 0.47 | 131.110 | |
| (3) | 63.999 | 0.47 | 136.167 | |
| BBa_J23115 + BBa_E0240 | (1) | 1.389 | 0.37 | 3.754 |
| (2) | 1.353 | 0.37 | 3.656 | |
| (3) | 1.370 | 0.33 | 4.151 | |
Fluorescence data is provided in relative fluorescence units and absorbance is unitless. Fluorescence values were calculated subtracting the average value of fluorescence of three samples of phosphate buffer (286.1) to the value given for each sample by the fluorometer.
For each set of samples, the mean and standard deviation for each quantity was calculated so they can be compared between them.
Table 2. Mean and standard deviation for each set of samples.
| Sample | Fluorescence | Optical density | Fluorescence / Optical density |
|---|---|---|---|
| Negative control | 1.065 ± 0.026 | 0.373 ± 0.021 | 2.857 ± 0.100 |
| BBa_I20260 | 4.385 ± 0.775 | 0.320 ± 0.053 | 13.684 ± 0.275 |
| BBa_J23101 + BBa_E0240 | 61.004 ± 3.346 | 0.457 ± 0.023 | 133.583 ± 2.530 |
| Bba_J23115 + BBa_E0240 | 1.370 ± 0.018 | 0.357 ± 0.023 | 3.854 ± 0.262 |
As it can be observed, all devices report fluorescence when compared to the negative control. The device which reports the highest expression of GFP is BBa_J23101+BBa_E0240, BBa_I20260 also shows fluorescence and BBa_J23115+BBa_E0240 presents the lowest value of fluorescence. It can be concluded that the BBa_J23101 promoter is much stronger than BBa_J23115.
