Difference between revisions of "Team:HUST-China/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.

• Provenance and release

  ①Individuals 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 safety

  What 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

• Instrument

  What 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-LUDOX
  photo_filter

  a.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.

  tune

  b.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_chart

  a.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_half

  b.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
  grain

  a.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℃
  

  fingerprint

  b.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_inclusive

  Transformation:
  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.

  fingerprint

  Cell 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