Team:ZJUT-China/InterLab
INTERLAB
Our team participated in the Fifth International InterLaboratory Measurement Study this year. The interlab study aims to solve the problem of reliability and repeatability in synthetic biology study. This year we were asked to measure green fluorescent protein, which is used as a measurement marker. We followed the experiment protocol strictly to make sure our data is valid. After two weeks lab work, we got the data we need after several attempts.
Calibration Protocol
Three calibration measurement were done by the calibration protocol, including an OD reference point at 600nm, one particle standard curve and one fluorescent standard curve.
(Plate Reader: Molecular Device, Spectramax M5)★ OD600 reference point ★
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 | 0.066 | 0.0285 |
| Replicate 2 | 0.059 | 0.029 |
| Replicate 3 | 0.068 | 0.026 |
| Replicate 4 | 0.064 | 0.028 |
| Arith. Mean | 0.064 | 0.028 |
| Corrected Abs600 | 0.036 | |
| Reference OD600 | OD600 | |
| OD600/Abs600 | 1.728 |
Fig1.Particle standard curve
Fig2.Particle standard curve (log_scale)
Fig3.Fluorescein standard curve
Fig4.Fluorescein standard curve (log_scale)
Cell measurement
Transformation
Transform Escherichia coli DH5α with these following plasmids:
| Device | Part Number | Location |
|---|---|---|
| Positive control | BBa_R0040 | Well 2D |
| Negative control | Ba_I20270 | Well 2B |
| Test Device 1 | BBa_J364000 | Well 2F |
| Test Device 2 | BBa_J364001 | Well 2H |
| Test Device 3 | BBa_J364002 | Well 2J |
| Test Device 4 | BBa_J364007 | Well 2L |
| Test Device 5 | BBa_J364008 | Well 2N |
| Test Device 6 | BBa_J364009 | Well 2P |
The transformation used E.coli DH5α competent cells bought from Tsingke Biological Technology company, and follow the steps in http://parts.igem.org/Help:2017_DNA_Distribution to use the DNA in the Distribution Kit.
Measurement
For this part of inter lab study, we followed the following protocols:
1.Grown 8 devices in incubator for 12 hrs at 37 ℃
2.Pick 2 colonies from each of plate and inocubate them on 10mL LB medium with Chloramphenicol. Grow the cells for 16-20hrs at 37°C and 180rpm.
3.Measure OD600nm of the overnight cultures and record the data, then dilute to target OD600nm = 0.02 in the falcon tubes.
4.Measure the OD600nm and Fl under the same condition as standard curve measurement and use the same 96 wells plates.
plate lay out
★ Fluorescence Raw Readings：★
★ Abs600 Raw Readings：★
★ uM Fluorescein / OD ★
★ Net Fluorescein a.u. ★
★ Net Abs 600 ★
Result of Raw Plate Reader Measurements
CFU Protocol
For this part of inter lab study, we followed the following protocol:
1.Pick two colonies from positive control device and negative control device, incubate overnight.
2.Prepare Starting Samples: Dilute the overnight cultures 1:8, measurement then dilute further to OD600nm = 0.1
Calculation:
Use (C1)(V1) = (C2)(V2) to calculate your dilutions
C1 is your starting OD600 C2 is your target OD600 of 0.1
V1 is the unknown volume in μL V2 is the final volume of 1000 μL
3.Check and make sure OD600 = 0.1
4.Dilute
5.Incubate at 37 degree Celsius overnight for 18-20 hrs. Then count colony number.
