We are very pleased to participate in the 5th iGEM team's fifth International Symposium on Synthetic Biology. Our team is involved in this research aimed at reducing laboratory-to-laboratory variability in fluorescence measurements by normalization to absolute cell count or colony forming units (CFU). In our case, we used a plate reader to measure OD and fluorescence. We additionally participated in flow cytometry protocol measurements.
Team:DLUT China/InterLab
Interlab
Fluorescence is widely used as a representative of promoter activity. Although this is an indirect measurement, fluorescent proteins such as green fluorescent protein (GFP) provide useful insights into expression levels and can be continuously monitored without destroying the cells.
First, measure three calibration protocols: use LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain conversion factor to convert absorbance (Abs600) to OD 600, use silica gel beads - microsphere suspension Obtain a particle standard curve,the fluorescein standard curve was measured with fluorescein at an excitation wavelength of 495 nm and an emission wavelength of this filter of 525 nm.
After making the standard curve, we successfully transformed the eight plasmids provided by the measurement committee and performed cell assays according to the protocol.
First, measure three calibration protocols: use LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain conversion factor to convert absorbance (Abs600) to OD 600, use silica gel beads - microsphere suspension Obtain a particle standard curve,the fluorescein standard curve was measured with fluorescein at an excitation wavelength of 495 nm and an emission wavelength of this filter of 525 nm.
After making the standard curve, we successfully transformed the eight plasmids provided by the measurement committee and performed cell assays according to the protocol.
3.1 Plasmid Used
Negative control:BBa-R0040
Possitive control:BBa-I20270
Test device1:BBa-J364000
Test device2:BBa-J364001
Test device3:BBa-J364002
Test device4:BBa-J364007
Test device5:BBa-J364008
Test device6:BBa-J364009
3.2 Strain usedPossitive control:BBa-I20270
Test device1:BBa-J364000
Test device2:BBa-J364001
Test device3:BBa-J364002
Test device4:BBa-J364007
Test device5:BBa-J364008
Test device6:BBa-J364009
Escherichia coli DH5a
3.3 Materials
1ml LUDOX CL-X
ddH2O
96 well plate,black with clear flat bottom
Clear plates
300 mL Silica beads - Microsphere suspension (provided in kit,4.7×10^8 microspheres)
Fluorescein
10ml 1×PBS pH 7.4-7.6
LB (Luria Bertani) liquid media
LB (Luria Bertani) solid media
Chloramphenicol (stock concentration 25 mg/ml dissolved in EtOH)
50 ml Falcon tube
1.5 ml tube
Ice bucket with ice
Micropipettes and tips
3.4 MachinesddH2O
96 well plate,black with clear flat bottom
Clear plates
300 mL Silica beads - Microsphere suspension (provided in kit,4.7×10^8 microspheres)
Fluorescein
10ml 1×PBS pH 7.4-7.6
LB (Luria Bertani) liquid media
LB (Luria Bertani) solid media
Chloramphenicol (stock concentration 25 mg/ml dissolved in EtOH)
50 ml Falcon tube
1.5 ml tube
Ice bucket with ice
Micropipettes and tips
BD FACSCanto Flow cytometer
SpectraMax M2 full wavelength microplate reader
Medifuge™ Small Benchtop Centrifuge
Eppendorf large Centrifuge
Constant temperature incubator
Constant temperature water bath
Vortex oscillator
3.5 MethodsSpectraMax M2 full wavelength microplate reader
Medifuge™ Small Benchtop Centrifuge
Eppendorf large Centrifuge
Constant temperature incubator
Constant temperature water bath
Vortex oscillator
Details
5 Discussion:
1. According to the fluorescence raw readings, at 0h, the fluorescence of Device 3 is weakest, which is similar to the negative control and the fluorescence of Device 4 is strongest, which increased 747% compared with the positive control. At 6h, the fluorescence raw reading data has the same law with the data at 0h, and the fluorescence of Device 4 has increased 632% compared with the positive control. The fluorescence of each group increased from 0h to 6h with the growth of bacteria. Abnormally, the fluorescence of the two bacteria of Device 5 is different.
2. As can be seen from the results, the number of colonies was 103 when diluted to 10-4, 102 when diluted to 10-5, and 101 when diluted to 10-6. According to the best counting principle, we select a plate with 50-200 colonies to count. For the specific values, please see the above results. The CFU of the positive control is calculated to be 7.58×107 (CFU/ml), and the CFU of the negative control is 1.01× 108 (CFU/ml). There is a significant difference between the two, indicating that the number of live bacteria per ml of the negative control in the 0h culture is greater than that of the positive control, and the results are also consistent with the above Abs results.
2. As can be seen from the results, the number of colonies was 103 when diluted to 10-4, 102 when diluted to 10-5, and 101 when diluted to 10-6. According to the best counting principle, we select a plate with 50-200 colonies to count. For the specific values, please see the above results. The CFU of the positive control is calculated to be 7.58×107 (CFU/ml), and the CFU of the negative control is 1.01× 108 (CFU/ml). There is a significant difference between the two, indicating that the number of live bacteria per ml of the negative control in the 0h culture is greater than that of the positive control, and the results are also consistent with the above Abs results.