Team:Washington/InterLab

Washington iGEM

InterLab


Overview

Since 2014, the iGem Measurement Committee has been searching for a means to make measurements for Green Fluorescent Protein repeatable anywhere. The InterLab study aims to allow the Measurement Committee explore ways to improve the measurement procedures for GFP that can be repeated in any lab anywhere.


For the fourth InterLab, teams were provided with highly detailed transformation and measurement protocols, as well as six test devices (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005) and both a positive (BBa_I20270) and a negative (BBa_R0040) control. Parts are located in the pSB1C3 plasmid backbone and carried chloramphenicol resistance. All plasmids were transformed into E.coli DH5-alpha cells


Methods and Materials

Plate reader: BioTek Synergy HIX

Abs OD600:
  • Wavelength (nm): 600
  • Measurements per data point: 8
  • Orbital Averaging: 3mm
  • Pathlength Correction: none
Fluorescence:
  • Excitation (nm): 485
  • Emission (nm): 528
  • Optics: Top
  • Gain: 75
  • Read speed: Normal
  • Read height: 1mm
  • Measurements per data point: 10

Materials
  • 1 mL LUDOX
  • dH₂O
  • 96 well plate (clear, flat-bottomed)
Methods:
  1. 100 μL of LUDOX-S40 from the InterLab Measurement Kit added into wells A1, B1, C1, and D1
  2. 100 μL of dH₂O added to wells A2, B2, C2, D2
  3. Absorbance at 600 nm taken for all samples in setup described above
  4. Data used to obtain correction factor

Materials:
  • Fluorescein
  • 10 mL 1xPBS (phosphate buffered saline)
  • 96 well plate (clear, flat-bottomed)
Methods:
  1. Fluorescein stock tube from InterLab Measurement Kit spun down to make sure pellet is at bottom of tube
  2. 2x fluorescein stock solution (100 μM) prepared by resuspending fluorescein in 1 mL of 1xPBS
  3. 500 μL of 2x fluorescein stock solution diluted with 500 μL of 1xPBS to make 1 mL of 50 μM (1x) fluorescein solution
  4. Serial dilutions of fluorescein:
    1. 100 μL of PBS added into wells A2, B2, C2, D2... A12, B12
    2. 200 μL of fluorescein 1x stock solution added to A1, B1, C1, D1
    3. 100 μL fluorescein stock solution transferred from A1 into A2
    4. A2 mixed by pipetting up and down, then 100 μL transferred to A3
    5. A3 mixed by pipetting up and down, then 100 μL transferred to A4
    6. A4 mixed by pipetting up and down, then 100 μL transferred to A5
    7. A5 mixed by pipetting up and down, then 100 μL transferred to A6
    8. A6 mixed by pipetting up and down, then 100 μL transferred to A7
    9. A8 mixed by pipetting up and down, then 100 μL transferred to A9
    10. A9 mixed by pipetting up and down, then 100 μL transferred to A10
    11. A10 mixed by pipetting up and down, then 100 μL transferred to A11
    12. A11 mixed by pipetting up and down, then 100 μL transferred to liquid waste
  5. Step 4 repeated for rows B-D
  6. Fluorescence for all samples measured using setup described above

Materials:
  • Competent Cells (Escherichia coli strain DH5 alpha)
  • LB media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 μg/mL)
  • 50 mL Falcon tubes covered in foil
  • Incubator at 37°C
  • Devices from InterLab Measurement Kit
    • Positive Control: BBa_I20270
    • Negative Control: BBa_R0040
    • Test Device 1: BBa_J364000
    • Test Device 2: BBa_J364001
    • Test Device 3: BBa_J364002
    • Test Device 4: BBa_J364003
    • Test Device 5: BBa_J364004
    • Test Device 6: BBa_J364005
Methods:
  1. Each of the plasmids listed above were located on Plate 6 of the distribution kit and resuspended in 10 μL of dH₂O
  2. Each device was transformed into 50 μL of competent cells
  3. Transformations were plated onto an agar plate with chloramphenicol and grown overnight
  4. On Day 2, 2 colonies were picked from each plate and inoculated in 10 mL of LB medium + Chloramphenicol in 50 mL Falcon tubes covered with foil. These cultures were grown overnight in an incubator at 37°C and 220 rpm.
  5. On Day 3, 100 μL of each culture was pipetted into a 96 well plate for an OD600 reading, which was then used for the Dilution Calculations in the Excel sheet provided by iGEM
  6. Each culture was diluted to a target OD of 0.02 following the preloading culture and media volumes calculated by the Dilution excel sheet (see OD Readings and Dilutions Calculations in the Results section below) in 10 mL of LB media + chloramphenicol in 50 mL falcon tubes wrapped in foil
  7. Cultures were placed in incubator at 37°C and 220 rpm 4 replicates of 100 μL samples were taken from each culture and held on ice at 0, 2, 4, and 6 hours of incubation before being placed in a 96 well plate for OD and fluorescence measurements using the setup described above.

Results





Dilution Calculations

Sample Abs600 Reading Volume of Preloading Culture (mL) Volume of Preloading Media (mL)
Positive Control (1) 0.608 0.3546 9.645
Positive Control (2) 0.618 0.3236 9.676
Negative Control (1) 0.621 0.3466 9.653
Positive Control (2) 0.613 0.3263 9.674
Device 1 (1) 0.806 0.2625 9.738
Device 1 (2) 0.788 0.2538 9.746
Device 2 (1) 0.618 0.3484 9.652
Device 2 (2) 0.453 0.4415 9.558
Device 3 (1) 0.622 0.3460 9.654
Device 3 (2) 0.609 0.3284 9.672
Device 4 (1) 0.602 0.3584 9.642
Device 4 (2) 0.598 0.3344 9.666
Device 5 (1) 0.624 0.3448 9.655
Device 5 (2) 0.594 0.3367 9.663
Device 6 (1) 0.574 0.3774 9.623
Device 6 (2) 0.534 0.3745 9.625
Media + Chl 0.044 10