Team:UFlorida/InterLab

InterLab Study

Overview

Directly comparing measurements is a challenge for many synthetic biology researchers. Fluorescent measurement is difficult for researchers to compare because it can be analyzed and reported in a variety of ways. Through the InterLab 2017 study, the iGEM Measurement Committee seeks to provide researchers with a standard for measuring expression of GFP in a plate reader.

iGEM Teams worldwide contributed to the InterLab study by transforming DH5a E. coli with six test devices (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, and BBa_J364005), as well as a positive (BBa_I20270), and negative control (BBa_R0040). Teams then measured the fluorescence of the bacteria in a plate reader over a 6-hour period.

Materials and Methods

Calibrations

OD 600

Materials:

  • 1ml LUDOX
  • H20
  • Cuvette

Methods

  1. Add 1 mL LUDOX into a cuvette
  2. Add 1 mL H2O into a separate cuvette
  3. Measure the absorbance at 600 nm of all samples
  4. Record the data
    5. Fluorescein Fluorescence Standard Curve

    Materials

    • fluorescein
    • 10ml 1xPBS
    • 96 well plate, black with flat, transparent/clear bottom

    Methods

    1. Prepare 2x fluorescein stock solution (100 uM) by resuspending fluorescein in 1 mL of 1xPBS
    2. Prepare 1mL of 50 uM (1x) fluorescein solution by diluting 500 uL of the 2x fluorescein stock solution with 500 uL of 1xPBS
    3. Conduct serial dilutions of fluorescein in the well plate
      1. 100 uL of PBS added into wells A2, B2, C2, D2... A12, B12
      2. 200 uL of fluorescein 1x stock solution added to A1, B1, C1, D1
      3. 100 uL fluorescein stock solution transferred from A1 into A2
      4. A2 mixed by pipetting up and down, then 100 uL transferred to A3
      5. A3 mixed by pipetting up and down, then 100 uL transferred to A4
      6. Continue through A11, DO NOT continue dilution into A12
    4. Step 3 repeated for rows B-D
    5. Fluorescence for all samples measured using the plate reader

Cell Measurements: Day 1

Materials
  • Competent cells (Escherichia coli strain DH5α)
  • LB media
  • Chloramphenicol
  • Incubator at 37°C
  • Devices
    • BBa_J364000
    • BBa_J364001
    • BBa_J364002
    • BBa_J364003
    • BBa_J364004
    • BBa_J364005
    • BBa_I20270
    • BBa_R0040
Methods

Cell Measurements: Day 2

Cell Measurements: Day 3

Results

★ ALERT!

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InterLab

Bronze Medal Criterion #4

Standard Tracks: Participate in the Interlab Measurement Study and/or improve the characterization of an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2017 part number range.

For teams participating in the InterLab study, all work must be shown on this page.