Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values.
Results
Competency
Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 108 to 6 ⋅ 108 cfu/µg. The transformation efficiency was 6.6 ⋅ 105 cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.
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 six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter.
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 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. 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
Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures below show the results.
Figure 1. Particle Standard CurveFigure 1. Particle Standard Curve (log)
Fluorescence Standard Curve
The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures below show the results.
Figure 3. Fluorescence Standard CurveFigure 4. Fluorescence Standard Curve (log)
Cell Measurements
Transformation was done according to the iGEM protocol [3]. After that, two colonies were picked from each plate and inoculated in LB medium, which contained chroramphenicol. Cells were incubated over night and samples were taken in the next morning and six hours after that. Plate reader protocol [4] has more detailed description about the process.
Figure 5. AbsorbanceFigure 6. Fluorescence
