Difference between revisions of "Team:NKU China/InterLab"
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Revision as of 14:42, 14 September 2017
Backgrounds
To ensure the biobricks generated in iGEM competitions are repeatable and reliable enough to be put into specific use,a robust measurement procedurehas been developed for green fluorescent protein (GFP) over the last three years by the Measurement Committee, through the InterLab study.Also,numbers of teams participate in this standardized measurement project.We’ve also tried our best to make our contribution.
Aim
By providing a protocol in details as well as forms helping unify the analyse of data,the Measurement Committee hopes to handle the problems emerged when establishing a baseline for replicability of fluorescence measurements and identifying likely key sources of error.So with the same devices,the same protocols and devices,teams all over the world try to measure the fluorescence the devices controlling to generate and study the distribution pattern of those results.
Results
Abs600 of cultures of 8 strains at selected time Fluorescence intensity(FI) of cultures of 8 strains at selected time FI/Abs600 of cultures of 8 strains at selected time
Methods
Cell Transformation
The following devices (from InterLab Measurement Kit) were transformed into Escherichia coli DH5α:
- 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
Transformation Selection
2 colonies from each of plate were picked and inoculated on 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
Cell preservation
200 µL of each of the 16 overnight cell cultures was mixed with 400 µL 50% glycerol and the mixture was preserved at -20℃.
Calibration
1.OD600reference point
◻ Add 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
◻ Add 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
◻ Measure absorbance 600 nm of all samples in all standard measurement modes in
instrument
◻ Record the data in the table below or in your notebook
◻ Record the result in the sheets provided.The results are as followed
| LUDOX-HS40 | H2O | |
|---|---|---|
| Replicate 1 | 0.040 | 0.040 |
| Replicate 2 | 0.040 | 0.030 |
| Replicate 3 | 0.040 | 0.030 |
| Replicate 4 | 0.050 | 0.040 |
| Arith. Mean | 0.043 | 0.035 |
| Corrected Abs600 | 0.007 | |
| Reference OD600 | 0.043 | |
| OD600/Abs600 | 5.667 |
2 Fluorescence standard curve
Prepare the fluorescein stock solution:
◻ Spin down fluorescein stock tube 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. [Note: it is important that the fluorescein is properly dissolved. To
check this, after the resuspension you should pipette up and down and examine the
solution in the pipette tip – if any particulates are visible in the pipette tip continue
to mix the solution until they disappear.]
◻ 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.)
Prepare the serial dilutions of fluorescein:
◻ Add 100 µl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
◻ 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
TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.
◻ Repeat dilution series for rows B, C, D
◻ Measure fluorescence of all samples in all standard measurement modes in instrument
◻ Record the data in your notebook
◻ record the result in the sheets provided.The results are as followed
Cell recovery
5µL of preservation mixture of each of the 16 colonies was inoculated in 5 mL LB medium (with 35 ng/µL chloramphenicol) in a 15 mL tube.Cells were incubated overnight for 15 h at 37℃ and 220 rpm.
| uM Fluorescein | 50.00 | 25 | 12.5 | 6.25 | 3.125 | 1.5625 | 0.78125 | 0.390625 | 0.1953125 | 0.09765625 | 0.048828125 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Replicate 1 | 593.078 | 261.146 | 315.981 | 184.531 | 125.520 | 78.074 | 51.338 | 30.925 | 31.293 | 16.106 | 10.933 | 7.308 |
| Replicate 2 | 591.904 | 350.723 | 312.492 | 130.295 | 99.755 | 88.922 | 34.262 | 19.690 | 14.543 | 11.504 | 11.469 | 9.180 |
| Replicate 3 | 557.482 | 342.179 | 221.832 | 123.956 | 63.827 | 40.323 | 26.030 | 15.974 | 11.867 | 11.831 | 10.830 | 7.003 |
| Replicate 4 | 537.689 | 337.536 | 253.889 | 138.396 | 131.001 | 52.293 | 32.527 | 20.655 | 13.578 | 11.150 | 9.702 | 8.835 |
| Arith. Mean | 570.038 | 322.896 | 276.048 | 144.294 | 105.026 | 64.903 | 36.040 | 21.811 | 17.820 | 12.648 | 10.576 | 8.082 |
| Arith. Std. Dev | 27.161 | 41.527 | 46.019 | 27.468 | 30.658 | 22.462 | 10.797 | 6.402 | 9.050 | 2.322 | 1.041 | 1.086 |
Cell growth,Sampling and assay
1.The plate reader was set to read OD600. 2.OD600of the overnight cultures was measured and the data shown bellow.
| target Abs600 | 0.02 |
| target volume (mL) | 12 |
| sample | Abs600 Reading | Volume of Preloading Culture | Volume of Preloading Media |
|---|---|---|---|
| positive control | 0.138 | 2.667 | 9.333 |
| negative control | 0.334 | 0.839 | 11.161 |
| device 1 | 0.270 | 1.081 | 10.919 |
| device 2 | 0.318 | 0.889 | 11.111 |
| device 3 | 0.341 | 0.819 | 11.181 |
| device 4 | 0.284 | 1.017 | 10.983 |
| device 5 | 0.296 | 0.968 | 11.032 |
| device 6 | 0.328 | 0.857 | 11.143 |
| media+chl | 0.048 |
3.The cultures were diluted to a target OD600 of 0.02 in 10 mL LB medium(with 35 ng/µL chloramphenicol) in 50 mL falcon tubes.
4.The cultures were incubated at 37℃ and 220 rpm.
5.100µL samples of the culture were taken at 0,1,2,3,4,5 and6 hours of incubation.
6.The sample were placed on ice.
7.Samples were laid out according to the following figure.
8.Data was recorded.
