Team:HUST-China/Model

Interlab

「Interlab」

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 measure the expression level of GFP, in order to help the iGEM community collect data about how reliable will these devices 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

    • ① Prepare your 96 well plate
    • ②Add 100 μl LUDOX 100 % into wells A1, B1, C1, D1
    • ③Add 100 μl of dH2O into A2, B2, C2, D2
    • ④Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
    • ⑤Import data into "Abs600" blue cells in provided Excel calibration sheet

    • B1. Protocol for FIuorescein Fluoresence 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
    • ① Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
    • ② Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1ml of 1xPBS
    • ③ Dilute the 2x fluorescein stock solution using 1xPBS to make a 1x fluorescein solution (final concentration is 50 μM).
    • ④ *Illustration of serial dilution samples in 96 well plate or cuvettes: value decreases by 2-fold with each column (50% in column 2, 25% in column 3, 12.5% in column 4, etc.)
    • ⑤Import data into "Abs600" blue cells in provided Excel calibration sheet
    • 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:
    • ① Pick 2 colonies from each of plate and inoculate each in 5-10 mL LB medium + Chloramphenicol (For antibiotic concentrations, please follow these guidelines:http://parts.igem.org/Help:Protocols/Antibiotic_Stocks).
    • ② Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
    • ③ With 8 total devices (including controls) and 2 colonies per device, you should have 16 cultures.
    • Cell growth, sampling, and assay
    • ① Set your instrument to read Abs600 (as OD calibration setting)
    • ② Measure Abs600 of the overnight cultures
    • ③ Import data into blue cells in Excel Dilultion Calculation sheets provided
    • ④ Dilute the cultures to a target Abs600 of 0.02 (see the volume of preloading culture and media in Excel Dilution Calculation sheets) in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube.
    • ⑤ Remove 500uL of each culture for your zero time point and hold these samples on ice. (You should have 16 sample tubes from the time=0 time point)
    • ⑥ Incubate the cultures at 37°C and 220 rpm.
    • ⑦ Remove 500uL samples of each culture after 2, 4 and 6 hours of growth. Keep samples on ice while completing the additional time points. You should have 16 sample tubes for each time point.
    • ⑧ Set up your four measurement plates: For colony #1 culture, pipet 100 uL samples into wells A, B, C and D of the row for that device. For colony #2, pipet 100uL samples into wells E, F, G, and H of the row for that device.
    • ⑨ Read your plates, taking care to use the exact same settings used for your fluorescein measurement.
    • The initial OD600 measurement of our overnight cultures.
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    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
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    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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    demo1

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    demo1

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    Acknowledgments: