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 using a spectrophotometer *Approved by the InterLab Committee*
  4. Record the data
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
  1. Locate the devices listed above in the distribution kit and resuspend them in 10uL of dH20
  2. Transform each device into 50uL of competent cells
  3. Plate the transformations onto LB plates with chloramphenicol and incubate overnight at 37C

Cell Measurements: Day 2

Materials
  • LB broth
  • Chloramphenicol
  • 16 50 ml Falcon tubes
  • Aluminum Foil
  • Transformed Cells
  • Inoculating Loops
Methods
  1. Two colonies were selected from each of plate and inoculated it in 5 mL LB medium + Chloramphenicol in separate 50 ml Falcon Tubes. The Falcon tubes were then covered tightly with foil. The cells were grown overnight (16 hours) at 37°C and 220 rpm.

Cell Measurements: Day 3

Materials
  • Overnight Cultures
  • Luria Broth
  • Chloramphenicol
  • 1.5 ml eppendorf tubes for sample storage
  • Ice bucket with ice
  • Pipettes
  • 96 well plate, black with flat, transparent/clear bottom
Methods
  1. 1 mL of each culture was pipetted into a cuvette for an OD600 reading, which was then used for the Dilution Calculations in the Excel sheet provided by iGEM
  2. Each culture was diluted to a target OD of 0.02 in 12 mL of LB media + chloramphenicol in 50 mL falcon tubes wrapped in foil
  3. Cultures were placed in incubator at 37ºC and 220 rpm
  4. 500 µL samples of the cultures were taken at 0, 2, 4, and 6 hours of incubation. (At each time point, a sample was taken from each of the 8 devices, two colonies per device, for a total of 16 samples per time point)
  5. At each time point, the OD and fluorescence of the samples were measured

Results