Reliable and repeatable measurement is a key component to synthetic biology. One of the big challenges in synthetic
biology measurement is that fluorescence data usually cannot be compared because it is reported in different
units or because different groups process data in different ways. We aim to improve the measurement tools available
to both the iGEM community and the synthetic biology community as a whole. Teams were given twelve test
devices of green fluorescent protein (GFP) under kit plate 6 and 7, as well as a positive and a negative control（their
location listed in figure 1）. We were asked to measure the fluorescent output of these samples using equipment
in our lab.
Figure 1. The locations of devices
The CPU_CHINA team measured these samples on our Multimode Reader and reported the results to iGEM HQ for further
analysis and comparison to other teams' results before deadline（figure 2）.
Figure 2. The microbiological operating instrument that we used
Interlab study protocols
→ OD 600 Reference point
1. Add 100 μl LUDOX into wells A1, B1, C1, D1 of 96 well plate
2. Add 100 μl of H 2 O into wells A2, B2, C2, D2 of 96 well plate.
3. Measure absorbance 600 nm of all samples in all standard measurement modes in Instrument.
4. Export the data in the desktop.
5. Import data into Excel ( OD600 reference point tab ) Sheet_1 provided
→ Protocol fluorescein fluorescence standard curvet
1. Prepare the fluorescein stock solution:
centrifuge the fluorescein stock tube at 3000 rpm for 1 min to make sure pellet is at the bottom of tube.
Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration
of fluorescein stock solution 50 μM (500μL of 2x fluorescein in 500 μL 1x PBS will make 1 mL of 50 μM (1x) fluorescein
solution.)
2. Prepare the serial dilutions of fluorescein:
Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12 of 96 well plate.
Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1.
Transfer 100 μl of fluorescein stock solution from A1 into A2.
Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste（figure 3）.
Repeat dilution series for rows B, C, D.
Measure fluorescence of all samples in all standard measurement modes in instrument.
Export the data on desktop.
Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided.
Figure 3. Overview of 96 well plate setup
→ Cell measurement protocol(figure 4)
Notes: The measurement protocol takes kit plate 6 as an example. It is the same protocol, when measuring the kit
plate 7.
Figure 4. Overview of the Cell measurement protocol
Day 1 : transform Escherichia coli DH5a with these following plasmids:
● Positive control
● Negative control
● Test Device 1: J23101+I13504
● Test Device 2: J23106+I13504
● Test Device 3: J23117+I13504
● Test Device 4: J23101.BCD2.E0040.B0015
● Test Device 5: J23106.BCD2.E0040.B0015
● Test Device 6: J23117.BCD2.E0040.B0015
Day 2 : pick colonies
● Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol.
● Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
Day 3 : Cell growth, sampling, and assay
● Set your instrument to read OD600 (as OD calibration setting)
● Measure OD600 of the overnight cultures
● Record data in your notebook
● Import data into Excel ( Dilution Calculation ) Sheet_1 provided
● Dilute the cultures to a target OD 600 of 0.02 (see the volume of preloading culture and media in Excel ( Dilution
Calculation ) Sheet_1) in 12 m l LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to
block light).
● Incubate the cultures at 37°C and 220 rpm.
● Take 500 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time point, you will take a
sample from each of the 8 devices, two colonies per device, for a total of 16 samples per time point)
● Place samples on ice.
● At the end of sampling point you need to measure your samples (OD600 and Fl 485/530 measurement), (figure 5 for
details).
● Export data on the desktop.
● Import data into Excel ( cell measurement tab ) Sheet_1 provided.
Figure 5. Overview of the samples’ locations in 96 well plate for each time point
Interlab study results
→ OD 600 Reference point
We used LUDOX-S40 as a single point reference to obtain a ratiometric conversion factor and transform our absorbance
data into the standard OD 600 measurement (table6).
Table 1. The data of OD600 measurement
→ fluorescein fluorescence standard curve
We prepared a dilution series of fluorescein in 4 replicates and measure the fluorescence in a 96 well plate in the
plate reader. By measuring these in all standard modes in the plate reader, We got a standard curve of fluorescence
for fluorescein concentration (table 2 and 3).
Table 2. The data of fluorescence measurement
Table 3. The fluorescence standard curve
→ Cell measurement
Hereafter we measured the Abs600 and fluorescence of all of the cell samples we took. We measured the fluorescence
with the same settings as the standard modes( table4 and 5 ).
Table 4. The fluorescence of all of the cell samples(kit plate 6)
Table 5. The fluorescence and OD600 of all of the cell samples at 6 h(kit plate 6)
DISCUSSION
The fluorescence per OD600 of the different devices increased with promoter strength. We compare the intensity of
the promoter of the devices by comparing the ratio of the fluorescence value to the OD600 value ratio between
the samples.
→ kit plate 6
The ratio of device 1 is significantly larger than positive control. device1 ratio than the device2 about 2.5 times.
device2 ratio than the device4 about 1.4 times. And the ratio of device4 is 5 times larger than device5. device
3 and 6 are basically the same size and are balanced with negative control(Table6).
In summary, the strength of the promoter from large to small are: device1, device2, device4, device5, device3
/ 6.
→ kit plate 7
The ratio of device 1 is significantly larger than positive control. device1 ratio than the device2 about 2 times.
device2 ratio than the device4 about 1.8 times. And the ratio of device4 is 6 times larger than device5. device
3 and 6 are basically the same size and are balanced with negative control(Table7).
In summary, the strength of the promoter from large to small are: device1, device2, device4, device5, device3
/ 6
Table 6. The fluorescein/OD600 of samples at 6 h(kit plate 6)
Table 7. The fluorescein/OD600 of samples at 6 h(kit plate 7)
