Measurements should be able to be repeated, but because every laboratory has their own practices, instruments and devices, it is sometimes challenging and results are not the same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. Escherichia coli DH5α was transformed with different Green Fluorescent Protein (GFP) plasmids and a plate reader was used to measure the values.
Results
Competency
The DH5α cells were made competent by using the iGEM Competent Cells protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected the transformation efficiency. Cell competency was measured by using the Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid backbone pSB1C3. The plasmid concentrations in the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to the iGEM Competent Cell Test Kit protocol [2]. According to the iGEM Competent Cell Test Kit protocol [2], competent cells should have an efficiency of 1.5 ⋅ 108 to 6 ⋅ 108 cfu/µg. The calculated transformation efficiency was 6.6 ⋅ 105 cfu/µg, which is very low compared to that. However, the transformations were still successful.
Table 1. Colonies
DNA concentration [pg/µl]
10
100
Plate 1
10
51
Plate 2
5
89
Plate 3
3
76
Average
6
72
Table 2. Transformation efficiency
DNA concentration [pg/µl]
10
100
Plate 1
1 ⋅ 106 cfu/µg
5.1 ⋅ 105 cfu/µg
Plate 2
5 ⋅ 105 cfu/µg
8.9 ⋅ 105 cfu/µg
Plate 3
3 ⋅ 105 cfu/µg
7.6 ⋅ 105 cfu/µg
Average
6 ⋅ 105 cfu/µg
7.2 ⋅ 105 cfu/µg
Parts
The transformation of E. Coli DH5α was performed with eight different plasmids. The test device plasmids contained GFP that had small structural differences, e.g. different promoters.
Table 3. Parts
Device
Part Number
Negative control
BBa_R0040
Positive control
BBa_I20270
Test Device 1
BBa_J364000
Test Device 2
BBa_J364001
Test Device 3
BBa_J364002
Test Device 4
BBa_J364007
Test Device 5
BBa_J364008
Test Device 6
BBa_J364009
OD600 Reference Point
Because the plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD600 to Abs600. LUDOX CL-X and sterile water were used in the experiment according to the iGEM InterLab Plate Reader Protocol [4]. LUDOX CL-X is a 45 % colloidal silica suspension. Table 4 shows the results.
Table 4. Calibration 1
LUDOX CL-X
ddH2O
Replicate 1
0.051
0.041
Replicate 2
0.051
0.036
Replicate 3
0.048
0.036
Replicate 4
0.060
0.038
Arith. Mean
0.053
0.038
Corrected Abs600
0.015
Reference OD600
0.063
OD600/Abs600
4.271
Particle Standard Curve
A microsphere suspension was used to construct the particle standard curve. The microspheres are the same scale as the cells and the optical properties are also similar with the cells that were used. A dilution series of the microspheres was made in order to obtain the relation between absorbance and the amount of cells. This was done according to the iGEM InterLab Plate Reader Protocol [4]. The results were used to estimate the numbers of cells in certain absorbances. The standard curves are shown in figures 1 and 2.
Figure 1. Particle Standard CurveFigure 1. Particle Standard Curve (log)
Fluorescence Standard Curve
The fluorescence values vary depending on the device and because the measured values are relative, depending on the measuring parameters used. Fluorescence standard curves make it possible to compare the fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentrations. Finally, fluorescence was measured by using a plate reader. The experiment was done according to the iGEM InterLab Plate Reader Protocol [4]. The standard curves are shown in figures 3 and 4.
Figure 3. Fluorescence Standard CurveFigure 4. Fluorescence Standard Curve (log)
Cell Cultivation and Measurements
Transformation was done according to the iGEM Transformation protocol [3]. After that, two colonies were picked from each plate and inoculated in LB medium, which contained chloramphenicol. The cells were incubated overnight after which they were diluted to 0.02 abs600. Samples were taken at 0h and 6h from the diluted cultures. The cells were incubated at 37 °C, 220 rpm between the time points. The iGEM InterLab Plate Reader Protocol [4] has a more detailed description about the process.
Figure 5. AbsorbanceFigure 6. Fluorescence
Colony Forming Units per 0.1 OD600E. Coli cultures
Dilution series was performed to estimate the amount of bacteria in culture. iGEM protocol [4] assumed that one bacterial forms one colony. Dilutions 3-5 were plated, and colonies were counted after 18 hours incubation. The results were multiplied by dilution factor. The mean was 13.042 ⋅ 105 CFU/mL in 0.1 OD600.
