Team:HUST-China/InterLab

Interlab

Interlab

Overview

One of the big challenges in synthetic biology is that measurements of fluorescence usually cannot be compared because they are reported in different units or because different groups process data in different ways. Interlab study aims to encourage iGEM teams together to explore a questions: " How close can the numbers be when fluorescence is measured all around the world?" They provided two protocols to measuring GFP fluorescence that will result in common, comparable units for teams to test out. We used plate reader assay to gain GFP fluorescence intensity data, hoping it would contribute to measurement institution.

Plate reader form

  •  Individuals responsible for conducting InterLab study

    Lin Di:create the devices

    Wangjie Liu:conduct measurements

    Zhangyu Cheng:process data

  • Corresponding email

    Lin Di:U201412522@hust.edu.cn

    Wangjie Liu:liuwangjie@hust.edu.cn

    Zhangyu Cheng:zhangyuzheng@hust.edu.cn

Chassis and Safety Information

  • What chassis did you use?

    Escherichia coli DH5alpha

  • What Biosafety Level is your chassis?

    BSL1

  • What PPE did you utilize during your experiments (from cloning through to measuring the devices)?

    Tianming gloves

    Songxinjiujiu labcoats

Calibration Protocol

  • What instrument did you use during your measurements?

    plate reader

  • Please provide the brand and model of your instrumen

    Flexstation 3

OD600 Reference Point

You will use LUDOX-S30 as a single point reference to obtain a ratiometric conversion factor to transform your absorbance data into a standard LUDOX Abs600 you must use the same cuvettes, plates and volumes (suggestion: use 100 uL for plate reader measurement and 1mL for spectrophotometer measurement) that you will use in your cell based assays. If using plates prepare a column of 4 wells with 100 uL 100% LUDOX and 4 wells containing 100 uL H2O. Repeat the measurement in all relevant modes used in your experiments (e.g. setting for orbital averaging). If using a cuvette you will only have enough material for a single measurement, but repeat the reading multiple times. Use the same cuvette to measure the reference with H2O (this value will be subtracted by the instrument to give the OD600 reading).

Instrument setting

  • Did you use pathlength correction during measurement?

    Yes

  • Did you use pathlength correction during measurement?

    6

  • Orbital averaging (mm)

    600

  • What temperature setting did you use during the measurement?

    22℃

Measurement steps

✔️ Prepare your 96 well plate or cuvettes

✔️ Add 100 μl LUDOX 100 % into wells A1, B1, C1, D1 (or 1 mL LUDOX 100% into a cuvette)

✔️ Add 100 μl of H2O into A2, B2, C2, D2 (or 1 mL H2O into a cuvette)

✔️ Measure absorbance 600 nm of all samples in all standard measurement modes in  instrument

✔️ Import data into "Abs600" blue cells in provided Excel calibration sheet

FITC Standard Curve

In this protocol, you will prepare a dilution series of FITC in 4 replicates and measure the fluorescence in a 96 well plate in your plate reader or individually in cuvettes in a fluorimeter. This will allow your measurements to be converted from arbitrary fluorescence into units of "Molecules of Equivalent Fluorescein" (MEFL).

Before beginning this protocol ensure that you are familiar with the GFP settings and measurement modes of your instrument. The settings that you use should be exactly the same ones that you will use when measuring your cells (if you change them you will not be able to use this standard curve). If you aren't absolutely certain which you will use, it can be a good idea to repeat the measurement a number of times with different settings. You will then have a series of standard curves to choose from without needing to redo this protocol. Most important, it is necessary to use a number of settings that affect the sensitivity (principally gain and/or slit width). Be sure to also consider other options (orbital averaging, top/bottom optics). As before, TURN OFF path length correction if available.

Instrument setting

  • 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?

    515nm

  • Emission wavelength

    518nm

  • Excitation wavelength

    485nm

  • Fluorescence reading

    Top optic

  • What temperature setting did you use during the measurement?

    22℃

Measurement work flow

  • Part 1: Prepare the FITC stock solution

    more details

    • ✔️ Spin down FITC stock tube to make sure pellet is at the bottom of tube.

    ✔️ Prepare 2x FITC stock solution (500 μM) by resuspending FITC in 1ml of 1x Phosphate Buffer Saline (PBS).

    ✔️ Incubate the solution at 42°C for 4 hours.it is important that the FITC is properly dissolved. To check this after the incubation period pipetted up and down – if any particulates are visible in the pipette tip continue to incubate overnight.

    ✔️ Dilute the 2x FITC stock solution in half to make a 1x FITC solution (final concentration is 250 μM).

    ✔️ Other: plate were wrapped in tinfoil

  • Part 2 (Plate Reader): Prepare the serial dilutions of FITC

    more details

    • ✔️ Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12  

    ✔️ Add 200 μl of FITC stock solution into A1, B1, C1, D1  

    ✔️ Transfer 100 μl of FITC stock solution from A1 into A2.  

    ✔️ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3  

    ✔️ Mix A3 by pipetting up and down 3x and transfer 100 μl into A4  

    ✔️ Mix A4 by pipetting up and down 3x and transfer 100 μl into A5  

    ✔️ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6  

    ✔️ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7  

    ✔️ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8  

    ✔️ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9  

    ✔️ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10  

    ✔️ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11  

    ✔️ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste  

    ✔️ TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.  

    ✔️ Repeat dilution series for rows B, C, D  

    ✔️ Measure fluorescence of all samples in all standard measurement modes in instrument  

    ✔️ Other:  the plate were wrapped in tinfoi

  •  Part 3. Measurement

    more details

    • ✔️ Measure fluorescence of all of your samples.

    ✔️ Import data into "Fluorescence" blue cells in provided Excel calibration sheet

    ✔️ Other:  the plate were wrapped in tinfoil

Cell Measurement protocol

Prior to performing the measurement on the cells you should perform the calibration measurements.

This will ensure that you understand the measurement process and that you can take the cell measurements under the same conditions.

Measurement work flow

  • Day 1 : Transformation

    more details

    • ✔️ Positive control

    ✔️ Negative control

    ✔️ Device 1: J23101+I13504

    ✔️ Device 2: J23106+I13504

    ✔️ Device 3: J23117+I13504

  • Day 2 : Cell growth

    more details

    • pick 2 colonies from each of plate and inoculate it on 5­10 mL LB medium + Chloramphenicol.For antibiotic concentrations, please follow these guidelines:

    Grow the cells overnight (16­18 hours) at 37°C and 220 rpm

    Continue: Did you set up biological replicates in duplicate?

    Yes

  •  Day 3 : Cell growth, sampling, and assay

    more details

    • ✔️ Set your instrument to read OD600 (as OD calibration setting)

    ✔️ Measure OD600 of the overnight cultures.Import data into blue cells in Excel (normalisation) sheets provided. Dilute the cultures to a target OD600 of 0.02 (see the volume of preloading culture and media in excel (normalisation) sheets) in 10 ml 0.5x TB medium + Chloramphenicol in 50 mL falcon tube (if using cuvettes, you can use 100 ml in a 500 ml shake flask).

    ✔️ Incubate the cultures at 37°C and 220 rpm.

    ✔️ Take 100 µL (1% of total volume) cultures at 0, 1, 2, 3, 4, 5, and 6 hours of incubation (if using cuvettes, remove 1 ml from 100 ml culture).

    ✔️ Other: didn’t place on ice

  •  What is the initial OD600 measurement of your overnight cultures?
    • Sample Abs600 reading replicate1 Abs600 reading replicate2
    Positive control 2.215 2.686
    Negative control 2.652 2.677
    Device 1 1.338 1.093
    Device 2 2.844 2.235
    Device 3 2.436 2.78

Measurement work flow

  •  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

Recommended 96­well plate layout for Abs600 and Fluorescence measurement

  • What temperature setting did you use during the measurement?

    • 22℃

Measurement 

✔️ Measure OD and fluorescence of all samples

✔️ Import data into blue cells in Excel (cell measurement) sheets provided

Feedback

  • Please rate your experience with filling in this InterLab Protocol form

    Very easy to fill in, no problems

Interlab Result

  • OD600 Reference Point

    LUDOX H2O
    Replicate 1 0.056 0.046
    Replicate 2 0.056 0.044
    Replicate 3 0.054 0.046
    Replicate 4 0.056 0.046
    Average 0.0555 0.0455
    Corrected Abs600 0.01
    Reference OD600 0.01475
    Correction factor 1.475

  • FITC Standard Curve

    Concentration(μM) 250 125 62.5 31.25 15.625 7.8125 3.90625 1.953125 0.9765625 0.48828125 0.244140625 0
    replicate 1 7485.3 5363.6 2708.5 1397.2 754.51 342.4 191.91 95.683 52.765 72.865 14.185 1.583
    replicate 2 7807.6 5113.2 2665.2 1504.2 711.56 367.4 219.19 133.42 79.839 46.925 32.849 1.25
    Replicate 3 7438.5 4882.6 2692.8 1357.4 697.04 416.86 203.97 115.47 70.608 41.723 24.908 1.247
    replicate 4 7465.6 4628.4 2735 1334.7 779.89 449.04 242.69 148.17 93.355 60.175 41.599 1.19
    Mean 7549.25 4996.95 2700.375 1398.375 735.75 393.925 214.44 123.18575 74.14175 55.422 28.38525 1.3175
    SD 173.2985959 314.5638833 29.20940203 75.13048094 48.03498274 21.89251623 22.69277915 17.03976196 13.98464477 11.66571359 0.17913961 38.22694251

  • Normalisation

    Target Abs600 0.02
    Target volume (mL) 10
    Sample Abs600 reading Volume of preloading culture Volume of preloading media
    Positive control 2.215 0.090293454 9.909706546
    Negative control 2.652 0.075414781 9.924585219
    Device 1 1.338 0.149476831 9.850523169
    Device 2 2.844 0.070323488 9.929676512
    Device 3 2.436 0.082101806 9.917898194
    media+chl 0

  • Cell measurement

    Abs600 raw data
    Positive control Negative control Device 1 Device 2 Device 3 Media+chloramphenico
    Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2
    0h 0.048 0.048 0.047 0.05 0.047 0.048 0.045 0.047 0.051 0.046 0.043 0.04
    1h 0.053 0.049 0.055 0.057 0.05 0.049 0.053 0.052 0.054 0.047 0.042 0.042
    2h 0.082 0.061 0.115 0.111 0.078 0.065 0.085 0.087 0.095 0.09 0.043 0.045
    3h 0.125 0.083 0.158 0.157 0.12 0.103 0.127 0.131 0.141 0.142 0.043 0.048
    4h 0.147 0.128 0.184 0.172 0.131 0.129 0.165 0.177 0.165 0.15 0.046 0.045
    5h 0.174 0.152 0.204 0.19 0.142 0.14 0.174 0.179 0.186 0.18 0.045 0.051
    6h 0.181 0.175 0.232 0.217 0.147 0.139 0.202 0.201 0.198 0.205 0.045 0.044
    blank average 0.044428571
    correction factor 1.475


    Blank substraction and correction
    Positive control Negative control Device 1 Device 2 Device 3
    Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2
    0h 0.005267857 0.005267857 0.003792857 0.008217857 0.003792857 0.005267857 0.000842857 0.003792857 0.009692857 0.002317857
    1h 0.012642857 0.006742857 0.015592857 0.018542857 0.008217857 0.006742857 0.012642857 0.011167857 0.014117857 0.003792857
    2h 0.055417857 0.024442857 0.104092857 0.098192857 0.049517857 0.030342857 0.059842857 0.062792857 0.074592857 0.067217857
    3h 0.118842857 0.056892857 0.167517857 0.166042857 0.111467857 0.086392857 0.121792857 0.127692857 0.142442857 0.143917857
    4h 0.151292857 0.123267857 0.205867857 0.188167857 0.127692857 0.124742857 0.177842857 0.195542857 0.177842857 0.155717857
    5h 0.191117857 0.158667857 0.235367857 0.214717857 0.143917857 0.140967857 0.191117857 0.198492857 0.208817857 0.199967857
    6h 0.201442857 0.192592857 0.276667857 0.254542857 0.151292857 0.139492857 0.232417857 0.230942857 0.226517857 0.236842857

  • Fluorescence

    fluorescence raw data
    Positive control Negative control Device 1 Device 2 Device 3 Media+chloramphenico
    Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2
    0h 70.75 91.107 37.804 38.349 107.44 88.548 129.64 94.325 41.256 37.93 17.461 6.76
    1h 99.681 120.89 39.562 45.615 153.37 112.18 168.27 106.55 42.129 30.503 33.988 44.527
    2h 123.78 141.9 52.606 45.69 307.89 227.9 205.69 150.37 45.29 49.248 47.65 47.978
    3h 149.72 173.17 53.785 49.219 422.7 360.56 232.76 194.08 51.81 51.502 46.508 46.803
    4h 187.37 221.49 50.071 50.211 328.91 418.19 282.35 217.86 69.467 62.137 50.421 46.895
    5h 208.23 236.42 52.361 52.002 330.99 348.14 310.41 232.15 58.31 73.28 56.005 56.005
    6h 210.31 243.34 53.019 52.659 347.21 334.45 345.18 256.27 63.317 58.397 53.707 59.18
    Blank average 43.84914286


    Blank substraction and correction
    Positive control Negative control Device 1 Device 2 Device 3
    Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2
    0h 0.005267857 0.005267857 0.003792857 0.008217857 0.003792857 0.005267857 0.000842857 0.003792857 0.009692857 0.002317857
    1h 0.012642857 0.006742857 0.015592857 0.018542857 0.008217857 0.006742857 0.012642857 0.011167857 0.014117857 0.003792857
    2h 0.055417857 0.024442857 0.104092857 0.098192857 0.049517857 0.030342857 0.059842857 0.062792857 0.074592857 0.067217857
    3h 0.118842857 0.056892857 0.167517857 0.166042857 0.111467857 0.086392857 0.121792857 0.127692857 0.142442857 0.143917857
    4h 0.151292857 0.123267857 0.205867857 0.188167857 0.127692857 0.124742857 0.177842857 0.195542857 0.177842857 0.155717857
    5h 0.191117857 0.158667857 0.235367857 0.214717857 0.143917857 0.140967857 0.191117857 0.198492857 0.208817857 0.199967857
    6h 0.201442857 0.192592857 0.276667857 0.254542857 0.151292857 0.139492857 0.232417857 0.230942857 0.226517857 0.236842857

  • Fl/Abs600

    blank substraction
    positive control negative control device 1 device 2 device 3
    replicate 1 replicate 2 replicate 1 replicate 2 replicate 1 replicate 2 replicate 1 replicate 2 replicate 1 replicate 2
    0h 26.90085714 47.25785714 -6.045142857 -5.500142857 63.59085714 44.69885714 85.79085714 50.47585714 -2.593142857 -5.919142857
    1h 55.83185714 77.04085714 -4.287142857 1.765857143 109.5208571 68.33085714 124.4208571 62.70085714 -1.720142857 -13.34614286
    2h 79.93085714 98.05085714 8.756857143 1.840857143 264.0408571 184.0508571 161.8408571 106.5208571 1.440857143 5.398857143
    3h 105.8708571 129.3208571 9.935857143 5.369857143 378.8508571 316.7108571 188.9108571 150.2308571 7.960857143 7.652857143
    4h 143.5208571 177.6408571 6.221857143 6.361857143 285.0608571 374.3408571 238.5008571 174.0108571 25.61785714 18.28785714
    5h 164.3808571 192.5708571 8.511857143 8.152857143 287.1408571 304.2908571 266.5608571 188.3008571 14.46085714 29.43085714
    6h 166.4608571 199.4908571 9.169857143 8.809857143 303.3608571 290.6008571 301.3308571 212.4208571 19.46785714 14.54785714


    Fl/Abs600
    Positive control Negative control Device 1 Device 2 Device 3
    Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2
    0h 5106.60339 8970.983051 -1593.822976 -669.2916123 16765.95104 8485.20678 101785.7627 13308.13559 -267.5313191 -2553.713405
    1h 4416.079096 11425.55085 -274.9427393 95.23112481 13327.17949 10133.81356 9841.19774 5614.403582 -121.8416393 -3518.757062
    2h 1442.3303477 4011.431911 84.12543745 18.74736306 5332.235124 6065.706215 2704.430652 1696.384939 19.31628842 80.31879284
    3h 890.8474576 2273.059636 59.31222684 32.34018756 3398.745314 3665.937991 1551.083221 1176.50165 55.88807542 53.17517433
    4h 948.629432 1441.096335 30.2225769 33.80947862 2232.394697 3000.900137 1341.076392 889.8860316 144.0477147 117.4422605
    5h 860.1020313 1213.672767 36.1640593 37.97009364 1995.171849 2158.583264 1394.74595 948.6530642 69.25105612 147.1779393
    6h 826.3428126 1035.816489 33.14391935 34.61050623 2005.12346 2083.266936 1296.504756 919.7983422 85.94402838 61.42409072