Team:ZJU-China/InterLab
Introduction
The InterLab study is developed by the Measurement Committee. Since synthetic biology, also been called engineering biology, is based on engineering thinking and methods, it values the reliability and repeatability of measurements. The InterLab study, involving teams and labs around the world，takes green fluorescent protein (gfp) as an example after one strict, detailed protocol to ensure the common, comparable units for measuring, which hopes to test the robustness by comparing the results from different labs.
This is the Fourth Measurement InterLab and our team participates in it. The InterLab this year tries to establish a GFP measurement protocol based on engineering principles, find out the similarity between fluorescence measured around the world and make gene expression more precise and reliable through tests of some RBS devices (BCDs). We are provided with materials for standard curves together with positive control, negative control and devices with different promoters and RBSs. Strictly following the requirements and protocol, we have obtained the relevant datas.
METHODS & MATERIALS
Transformation
Transform Escherichia coli DH5α 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
Resuspend DNA in selected wells in the Distribution Kit with 10µL ddH20. Thaw competent cells on ice. Pipette 25µL of competent cells into 1.5mL tube per transformation and add 2µL of resuspended DNA into it. Incubate on ice for 30min. Heat shock tubes at 42°C for 90 sec. Then incubate on ice for 5min.
Add 200µL SOC media with Chloramphenicol(1000×) to each transformation. Incubate at 37°C for 1 hours, shaking at 200-300rpm.
Pipette 100µL of each transformation onto LB plates(Chloramphenicol, 1000×). Spread with sterilized spreader. Incubate transformations overnight (14-18hr) at 37°C.
Colonies Selection
Pick 2 single colonies from each of plate and inoculate it on 5-10 mL LB medium with Chloramphenicol(1000×). Grow the cells overnight (16-18 hours) at 37°C and 220 rpm. Check them under the fluorescence microscope before further experiments.
Calibration
We used the plate reader Synergy Neo2 for all the measurements and we used black 96 well plates with flat, transparent bottom.
OD600 Reference Point
Add 100 µl LUDOX into wells A1, B1, C1, D1 and 100 µl of H2O into wells A2, B2, C2, D2. Measure absorbance 600nm of all samples in all standard measurement modes in instrument, pathlength correction was turned off. The temperature setting was 24℃. Record the data.
Fluorescein Fluorescence Standard Curve
Spin down fluorescein stock 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.
Prepare the serial dilutions of fluorescein as shown below. Set 4 copies.
Fig.1 dilution[1]
Measure the plate in plate reader, the excitation filter was set to 485nm/5nm and the emission filter was set to 530nm/30nm. Pathlength correction was turned off. The gain setting was 70. Fluorescence was from the top. The temperature setting was 24℃. Record the data.
Cell Measurement
Measure OD600 of the overnight cultures prepared after colony selection, then dilute the culture to a target OD600 of 0.02 in LB medium + Chloramphenicol with the help of Excel(Dilution Calculation) sheet. Incubate the cultures at 37°C and 220 rpm. Take 500 µL samples of the cultures from each of the 8 devices, two colonies per device, at 0, 2, 4, and 6 hours of incubation and add them into 96 well plates as shown below. Place samples on ice before measurements
Fig.2 loading samples[1]
Measure the samples (OD and Fl measurement). The cell measurement was under the same condition with fluorescein fluorescence standard curve and used the same plate.
Results
OD600 Reference point
| LUDOX-HS40 | H2O | |
|---|---|---|
| Replicate 1 | 0.05 | 0.035 |
| Replicate 2 | 0.049 | 0.037 |
| Replicate 3 | 0.049 | 0.039 |
| Replicate 4 | 0.049 | 0.039 |
| Arith. Mean | 0.04925 | 0.0375 |
| Corrected Abs600 | 0.01175 | |
| Reference OD600 | 0.0425 | |
| OD600/Abs600 | 3.617021277 |
Table.OD600 reference point
Fluorescein Fluorescence Standard Curve
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Fig.3 standard curve 1 | Fig.4.standard curve 2
Discussion
Fig.5 Abs 600 measurement
Abs600 of the samples changed over time as shown, in which the data of each sample is the average of 4 replicates. Abs600 of the samples reflected its concentration, which could show the growth of bacteria. As the picture shown, the growth rate of bacteria generally got slower as time went by, this is probably because at the beginning of the measurement, bacteria was in the later period of logarithmic phase and the resources were not that sufficient, inhibiting the growth. Several samples appeared to be different, such as colony 1 of device 4, which seemed to be in the logarithmic phase and colony 2 of device 1, whose concentration went down after 2 hours' incubation, perhaps low activity resulting in massive death.
In addition, growth curves of each colony were different. This may be caused by different initial concentration of samples after dilution, which led the samples be in different growing statuses. We will mention it later.
Fig.6 relative fluoresence measurement | Fig.7 absolute fluorescence plot
The fluorescence differed obviously. The absolute fluorescence and relative fluorescence of device 3, device 5 and device 6 were relatively low, for which their promoters and RBSs might account. For two replicates of device 1, their fluorescence differed greatly and the same as growth, making it hard to compare. Either operational error or colony selection would affect.
Furthermore, the tendence of absolute fluorescence turned out to rise and fall, mostly going down as time went by. We thought this was because the protein expression varied in different growth phase and growth of bacteria changed over time.
Reflection
Some of our operations might affect the results, which can be improved.
Our lab do not have plate reader, so we borrowed one from other lab. We cultured bacteria and loaded samples in our lab and then took them to the other lab for measurement. Since it took some time to go back and forth, our actual measurement took longer than expectation, so the measurement postponed a little. Though samples were laid on ice, the bacteria was still growing, making the real measurement time different from the demand. Similarly, the cultures settled in the process, leading to the deviation of the concentration after dilution. As a result, the comparability between each sample was affected.
By the way, the first time we did the OD600 Reference Point measurement, we put the 96 well plate containing LUDOX on ice. Then it turned out that the OD600 of LUDOX was lower than water. Maybe LUDOX denatured in the cold environment on ice according to the official warning. So perhaps it is better not to put LUDOX on ice or anywhere like that.
Feedback
After the InterLab Study measurement, we have something to say about the provided protocol, which goes on like this:
Advantages
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Improvements
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We sincerely hope the InterLab Study this year will success and hope the InterLab Study next year will go well.
Reference
[1] https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf
[2] https://2017.igem.org/Competition/InterLab_Study
[3] https://2017.igem.org/Competition/InterLab_Study/Plate_Reader
