Team:ZJUT-China/InterLab
Team:ZJUT-China
Interlab
Our team has participated in the Fifth International InterLaboratory Measurement Study this year. The Interlab study aims on solving the problem of reliability and repeatability of synthetic biology study. This year we were asked to measure green fluorescent protein, which is usually used as a measurement marker. We followed the experiment protocol strictly to make sure our data are valid. After two weeks' lab work, we got the data we needed after several attempts.
Calibration Protocol
Three calibration measurements were done by following the calibration protocol, including an OD reference point at 600nm, one particle standard curve and one fluorescence standard curve.
★ 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 |
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 was bought from Tsingke Biological Technology company, and we followed 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 ODnm = 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.
★ Fluorescence Raw Readings：★
★ Abs600 Raw Readings：★
★ uM Fluorescein / OD ★
★ Net Fluorescein a.u. ★
★ Net Abs 600 ★
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 samples at 37 degree Celsius overnight for 18-20 hrs. Then count colony numbers.
Disscussion & Feedback
After the lab work, we had a discussion about the interlab study we did this year and now we have some feedbacks.
1.The interlab study protocol is very detailed and clear, we followed almost every step. But in some attempts, we tried to dilute the cultures in 96 wells plate, which is easier and quicker. So we do think improvements could be made to adjust the protocol.
2.The Excel sheets provide us an easy way to process our data. However, we still wish to know the meaning behind every function. It took us some time to search online, things would be easier if there is a guide.
3.The protocol tells us some principles of the experiment and it’s unbelievably helpful for us to complete the whole lnterlab study.
4.The kit needs a handbook to help those teams who participate in interlab study for the first time.
Collaboration
On August 8, 2018. We had a meeting with team jiangnan_China, team DLUT-China and team LZU-China in Jiangsu Normal University. By then we have already finished the interlab study successfully, but team jiangnan_China and team LZU-China met some difficulties. We provided them with some guidance and advice.
