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/test36
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.
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
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
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
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
MethodsSpectraMax M2 full wavelength microplate reader
Medifuge™ Small Benchtop Centrifuge
Eppendorf large Centrifuge
Constant temperature incubator
Constant temperature water bath
Vortex oscillator
Details
Discussion:
1. According to the Abs results at each time point 0 hours and 6 hours, the cell concentration of Device 1 is significantly lower than that of other devices.
2. According to the fluorescence raw readings, the fluorescence of Device 3 is weakest, the fluorescence of Device 4 is strongest. And the fluorescence of each group increased from 0h to 6 h, abnormally, the fluorescence of the two bacteria of Device 5 was different.
3. There is significant difference in plasmid conversion rate between negative control (device 1) and positive control (device 2).
2. According to the fluorescence raw readings, the fluorescence of Device 3 is weakest, the fluorescence of Device 4 is strongest. And the fluorescence of each group increased from 0h to 6 h, abnormally, the fluorescence of the two bacteria of Device 5 was different.
3. There is significant difference in plasmid conversion rate between negative control (device 1) and positive control (device 2).