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.

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.

Table 2

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

Figure 1