Team:DTU-Denmark/InterLab
InterLab Study
Standardization and reproducibility are two integral parts of synthetic biology. The InterLab study aims to reduce the variability of measurements made between laboratories by providing standardized protocols and data analysis procedures. Our team participated in the 4th International InterLab Study and submitted our results for further analysis. A summary of our results is described below.
Experimental Design
The teams participating in the 2017 InterLab study was provided a range of different GFP expressing devices to test. The devices along with their constituent parts are shown in table 1.
| Test Device # | Part Name | Used Promoter | Used RBS |
|---|---|---|---|
| Device 1 | BBa_J364000 | BBa_J23101 | BBa_B0034 |
| Device 2 | BBa_J364001 | BBa_J23106 | BBa_B0034 |
| Device 3 | BBa_J364002 | BBa_J23117 | BBa_B0034 |
| Device 4 | BBa_J364003 | BBa_J23101 | BBa_J364100 |
| Device 5 | BBa_J364004 | BBa_J23106 | BBa_J364100 |
| Device 6 | BBa_J364005 | BBa_J23117 | BBa_J364100 |
| Positive Control | BBa_I20270 | BBa_J23151 | BBa_B0032 |
| Negative Control | BBa_R0040 | BBa_R0040 | None |
Results
We successfully transformed cells with all the test devices shown in table 1, except test device 1 (BBa_J364000). The transformed cells were inoculated in liquid LB-CAM media overnight prior to testing. The plates were all analysed using a Cytation 5 plate reader (BioTek Instruments Inc.), and the plates used were all clear bottomed, white sided 96 well plates.
Calibration
The instrument used in the InterLab study was calibrated to adjust for measurement differences between plate readers from different laboratories. Furthermore, the calibration served to compensate for the light path not being 1 cm when measuring absorbance through a well plate. The calibration was done by loading the plate reader with a clear bottom 96 well plate containing wells with 100 uL LUDOX-S40 solution. The absorbance of the LUDOX-S40 was measured at 600 nm (A_600), and compared to the absorbance of a similar volume of dH2O. Based on the readings, a conversion factor of 4.25 could be calculated. Thus, for our experimental setup, the raw measurements of A_600 should be multiplied with the conversion factor in order to calculate the corresponding standard OD_600 value. In order to convert measured fluorescence readings to a concentration of expressed GFP, we constructed a standard curve by serial dilution of a 1 x fluorescein stock solution. The mean of the measured fluorescence for each concentration is described in table 2. The data from table 2 is plotted as Figure 1 to display the standard curve derived from the fluorescent measurements.
| μM | 12.5 | 6.2 | 3.1 | 1.56 | 0.78 | 0.39 | 0.19 | 0.09 | 0.04 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|
| Mean Fluorescens | 8172662 | 4422954 | 2335681 | 1168514 | 608365.3 | 304152.5 | 136802 | 70513 | 35051.25 | 253.75 |
The standard curve has an R^2 value of 0.9979 when assuming linearity. The high R^2 value means that it is be possible to calculate a concentration of GFP from absorbance readings with an acceptable accuracy.
Measurement
The transformants containing the test devices were inoculated into fresh LB CAM media, and incubated for 6 hours at 37C, with samples being taken from the culture every 2 hours. Fluorescence was measured with excitation at 485 nm, and emission at 530 nm (530/30 filter). Using the fluorescence readings, the concentration of expressed GFP/cell could be calculated from the measured optical density. This yielded the results shown in Figure 2.
Discussion
Our team was not able to measure fluorescence for the standard curve at concentrations of fluorescein above 12.5 uM, as the two highest concentrations of GFP yielded absorbance readings too high for the plate reader to measure. Despite the two missing data points, this standard curve was still submitted for the InterLab study, as the sensitivity setup used to create this particular standard curve proved to be the best setup for measuring our test devices. The team was not able to transform the Test Device 1 from the InterLab study, and we therefore have no real data for the expression of this device.
When looking at the expressed GFP per cell in Figure 2, it becomes apparent that both test device 2 and test device 4 had a high expression of GFP, outperforming that of the positive control after 4 hours. When comparing test device 2 and 5, it is clear that test device 2 has a higher expression of GFP, which could indicate that the BBa_B0034 RBS is stronger than the BBa_J364100 RBS. Test device 3 and test device 6 both had an expression of GFP similar to that of the negative control. Knowing that the BBa_J23117 promoter is relatively weak, this result was expected.
