Introduction
It is significant for synthetic biology to develop a reliable and repeatable measurement, the same to all the other engineering disciplines. We HUST-China have volunteered to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable by measuring the expression level of GFP, in order to help the iGEM community collect data about how reliable these devices will turn out to be in labs around the world.
Provenance and Release
①Individuals responsible for conducting InterLab study
Individuals |
Interlab Part |
Kangyuan Yu, Haibo Huang, Ziyang Xiao, Shaofeng Liao |
created the devices |
Efan Wang, Long Cheng, HuiPing Shi |
conducted the measurements |
Efan Wang |
processed the data |
②Corresponding email
Individuals |
Emails |
Efan Wang |
erfan@hust.edu.cn |
Kangyuan Yu |
985930862@qq.com |
Haibo Huang |
u201512127@hust.edu.cn |
Ziyang Xiao |
372657289@qq.com |
Shaofeng Liao |
15827233830@qq.com |
HuiPing Shi |
172295915@qq.com |
Long Cheng |
u201512127@hust.edu.cn |
Chassis and Safety
What chassis did you use?
Escherichia coli DH5alpha
What Biosafety Level is your chassis?
BSL1
What PPE did you utilize during your experiments?
Tianming gloves
Songxinjiujiu labcoats
Instrument
What instrument did you use during your measurements?
Plate reader
Please provide the brand and model of your instrument.
Flexstation 3
Calibration Protocol
A1. Protocol for Optical Density (OD600) Standard Measurement
Did you use pathlength correction during measurement?
Yes
Number of flashes per well
6
Orbital averaging (mm)
600
What temperature setting did you use during the measurement?
22℃
What type of 96-well plate did you use?
Black plate (preferred)
Did your plate have flat-bottomed or round-bottomed wells?
Flat
A2. Measurement Steps
B1. Protocol for Fluorescence in fluorescence standard curve
Did you use pathlength correction during measurement?
Yes
Number of flashes per well
6
What gain setting did you use?
Automatic
If you used a filter, what light wavelengths did it pass?
530nm
Emission wavelength
530nm
Excitation wavelength
485nm
Fluorescence reading
Bottom optic
What type of 96-well plate did you use?
Black plate (preferred)
Did your plate have flat-bottomed or round-bottomed wells?
Flat
What temperature setting did you use during the measurement?
22℃
B2. Measurement Steps
Part 1: Prepare the Fluorescein stock solution
Measurement work flow:
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Part 2: Prepare the serial dilutions of Fluorescein
①Add 100 μl of 1xPBS into wells A2, B2, C2, D2....A12, B12, C12, D12.
②Add 200 μl of 1x Fluorescein stock solution into A1, B1, C1, D1.
③ Transfer 100 μl of Fluorescein stock solution from A1 into A2.
④ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3.
⑤ Mix A3 by pipetting up and down 3x and transfer 100 μl into A4.
⑥ Mix A4 by pipetting up and down 3x and transfer 100 μl into A5.
⑦ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6.
⑧ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7.
⑨ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8.
⑩ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9.
⑪ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.
⑫ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.
⑬ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
⑭ Take care not to continue serial dilution into column 12.
⑮ Repeat dilution series for rows B, C, D.
⑯ Measure fluorescence of all samples in all standard measurement modes in instrument.
Cell Culture Setup and Measurement
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Transformation:
- 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
Cell growth:
Cell growth, sampling, and assay
The initial OD600 measurement of our overnight cultures.
What type of media did you use for this step?
Luria Bertani
What type of vessel or container did you use to grow your cells?
50 ml Falcon tube
What temperature setting did you use during the measurement?
22℃
What type of 96-well plates did you use?
Black plates with transparent/clear bottom (preferred)
Flat
Measurement
- Measure OD and fluorescence of all samples
- Import data into blue cells in Excel (cell measurement) sheets provided
Suggested Plate Layout for 96-well Plate
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Interlab Resultn
OD600 Reference Point
Fluorescence standard curve
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NOTE: 50uM Sample exceeds the range of measurements
OD600 Reference Point
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Final scaling level determined from medium-high points likely to be less impacted by saturation or pipetting error.
If needed, you can shift which points are used, but it is likely better to correct instrument settings and protocol.
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