Team:HUST-China/InterLab
Interlab
Introduction
This year, we HUST-China have volunteered to iGEM's fifth International InterLaboratory Measurement Study, in order to help the
iGEM community collect data about how reliable will devices turn out to be in labs around the world.
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Provenance and release①Individuals responsible for conducting InterLab study
individuals Interlab parts Ziyang Xiao Created devices Ziyang Xiao, Haotian Ren, Yan Chen, Hao Qiu Conducted the measurements Ziyang Xiao Processed the data ②Corresponding email
individuals emails Ziyang Xiao u201612166@hust.edu.cn Yan Chen u201713204@hust.edu.cn Haotian Ren u201712893@hust.edu.cn Hao Qiu u201713109@hust.edu.cn Chassis and safetyWhat chassis did you use? Escherichia coli DH5α What Biosafety Level is your chassis? BSL1 What PPE did you utilize during your experiments? Tianming gloves Songxinjiujiu labcoats
InstrumentWhat instrument did you use during your measurements? plate reader Please provide the brand and model of your instrument. Flexstation 3
Calibration 1：OD600 reference point-LUDOXphoto_filtera.Protocol
Use pathlength correction Number of flashes per well：6 Orbital averaging (nm)：600 Temperature setting：22℃ Type of 96-well plate：Black plate (preferred) Our plates have flat-bottomed wells.
tuneb.Measurement Steps
Add 100ul LUDOX into wells A1, B1, C1,D1 Add 100ul ddH2O into wells A2, B2, C2,D2 Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements Record the data in the table below or in your notebook data into Excel sheet provided (OD600 reference point tab)
Result
Calibration 2：Particle standard curve-microsphere bubble_charta.Protocol
Use pathlength correction Number of flashes per well：6 Orbital averaging (nm)：600 Temperature setting：22℃ Type of 96-well plate：Black plate (preferred) Our plates have flat-bottomed wells.
star_halfb.Measurement Steps
Prepare the Microsphere Stock Solution: ❏Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex ❏vigorously for 30 seconds. ❏Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube ❏Add 904 μL of ddH2O to the microspheres ❏Vortex well. This is your Microsphere Stock Solution.
Prepare the serial dilution of Microspheres:
polymer❏Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12 ❏Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds ❏Immediately add 200 μl of microspheres stock solution into A1 ❏Transfer 100 μl of microsphere 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...
spa❏ 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 ❏ Repeat dilution series for rows B, C, D ❏Re-Mix each row of your plate immediately before putting in the plate reader! Take care to mix gently and avoid creating bubbles on the surface of the liquid. ❏ Measure Abs600 of all samples in instrument ❏ Record the data in your notebook ❏ Import data into Excel sheet provided (particle standard curve tab)
Result
Calibration 3：Fluorescence standard curve – Fluorescein
graina.Protocol
Use pathlength correction Number of flashes per well： 6 Gain setting: Automatic It passed 530nm when we used a filter. Emission wavelength: 525nm Excitation wavelength: 485nm Fluorescence reading: Bottom optic Type of 96-well plate： Black plate (preferred) Our plates have flat-bottomed wells. Temperature setting: 22℃
fingerprintb.Measurement Steps
Prepare the fluorescein stock solution: ❏ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. ❏ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. ❏ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS
Prepare the serial dilutions of fluorescein:
flare❏Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12 ❏ Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1 ❏ Transfer 100 μl of fluorescein 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...
extension❏ 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 ❏ Repeat dilution series for rows B, C, D ❏ Measure fluorescence of all samples in instrument ❏ Record the data in your notebook ❏ Import data into Excel sheet provided(fluorescein standard curve tab)
Result
Cell culture setup and meaturement
all_inclusiveTransformation: Negative control BBa_R0040 Kit Plate 7 Well 2D Positive control BBa_I20270 Kit Plate 7 Well 2B Test Device 1 BBa_J364000 Kit Plate 7 Well 2F Test Device 2 BBa_J364001 Kit Plate 7 Well 2H Test Device 3 BBa_J364002 Kit Plate 7 Well 2J Test Device 4 BBa_J364007 Kit Plate 7 Well 2L Test Device 5 BBa_J364008 Kit Plate 7 Well 2N Test Device 6 BBa_J364009 Kit Plate 7 Well 2P
Cell grow: Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
fingerprintCell growth, sampling, and assay Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor) Measure Abs600 of these 1:10 diluted cultures Record the data in your notebook Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. Place the samples on ice. Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours. Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice. At the end of sampling point you need to measure your samples (Abs600 and fluorescence measurement) Record data in notebook Import data into Excel sheet provided (fluorescence measurement tab)
The initial OD600 measurement of our overnight cultures
Sample Abs600 reading replicate1 Abs600 reading replicate2 Negative control 0.106 0.104 Positive control 0.107 0.108 Device1 0.0937 0.108 Device2 0.119 0.105 Device3 0.106 0.116 Device4 0.109 0.104 Device5 0.106 0.104 Device6 0.104 0.109 Negative control 0.106 0.104 hdr_weak Type of media we used for this step: Luria Bertani
Type of vessel or container we used to grow cells: 50 ml Falcon tube
Temperature setting:22℃
Type of 96-well plates: Black plates with transparent/clear bottom (preferred),flat
Measurement: Measure OD and fluorescence of all samples
Suggested Plate Layout for 96-well Plate
Result
Counting colony-forming units (CFUs) from the sample.
This measurement, related to cell concentration of the culture, can be used to calibrate OD600 to colony forming unit (CFU) counts.
We measured and dilute our overnight cultures to OD600=0.1, and we checked the OD600 to ensure we have diluted to 0.1.Also,we did this in triplicate for each culture.
