Team:Harvard/InterLab

Harvard OPTI POLY

InterLab

Overview

Difficulty in taking reliable and reproducible measurements remains a key obstacle in establishing synthetic biology as an engineering discipline. The InterLab Study is part of the Measurement Committee’s continuing effort to develop a standard measurement procedure for green fluorescent protein (GFP). Despite being one of the most commonly used markers in synthetic biology, labs often resort to making relative comparisons, which makes it difficult for labs to share and data and/or constructs.

For the fourth installment of the InterLab, iGEM hopes to establish a GFP measurement protocol that can be used to produce comparable GFP measurements on different plate readers. In addition, iGEM teams from around the world have tested RBS devices (BCDs) intended to increase the precision and reliability of gene expression. The parts used include six test devices (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005), as well as a positive (BBa_I20270) and negative (BBa_R0040) control. All parts are located in the pSB1C3 plasmid and carry chloramphenicol resistance.

Methods and Materials

Plate Reader Setup

Materials
  • Plate Reader
Methods

OD600 Reference Point

Materials
  • 1 mL LUDOX
  • H2O
  • 96 well plate
Methods
  1. 100 uL of LUDOX-S40 from the InterLab Measurement Kit added into wells A1, B1, C1, and D1
  2. 100 uL 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

Protocol Fluorescein Fluorescence Standard Curve

Materials
  • Fluorescein
  • 10 mL 1xPBS (phosphate buffered saline)
  • 96 well plate
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 uM) prepared by resuspending fluorescein in 1 mL of 1xPBS
  3. 500 uL of 2x fluorescein stock solution diluted with 500 uL of 1xPBS to make 1mL of 50 uM (1x) fluorescein solution
  4. Serial dilutions of fluorescein:
    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. A4 mixed by pipetting up and down, then 100 uL transferred to A5
    7. A5 mixed by pipetting up and down, then 100 uL transferred to A6
    8. A6 mixed by pipetting up and down, then 100 uL transferred to A7
    9. A8 mixed by pipetting up and down, then 100 uL transferred to A9
    10. A9 mixed by pipetting up and down, then 100 uL transferred to A10
    11. A10 mixed by pipetting up and down, then 100 uL transferred to A11
    12. A11 mixed by pipetting up and down, then 100 uL transferred to liquid waste
  5. Step 4 repeated for rows B-D
  6. Fluorescence for all samples measured using setup described above

Cell Measurements

Materials
  • Competent Cells (Escherichia coli strain DH5 alpha
  • LB media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 ug/mL)
  • 50 mL Falcon tubes covered in foil
  • Incubator at 37ºC
  • Devices from InterLab Measurement Kit
    • Positive Control
    • Negative Control
    • Test Device 1: J23101 + I13504
    • Test Device 2: J23106 + I13504
    • Test Device 3: J23117 + I13504
    • Test Device 4: J23101.BCD2.E0040.B0015
    • Test Device 5: J23106.BCD2.E0040.B0015
    • Test Device 6: J23117.BCD2.E0040.B0015
Methods
  1. Each of the plasmids listed above were located on Plate 7 of the distribution kit and resuspended in 10 uL of dH2O
  2. Each device was transformed into 10 uL of competent cells (see here for the full transformation protocol)
  3. Transformations were spot plated onto an agar plate with chloramphenicol and grown overnight(see here for the full spot plating protocol)
  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 37ºC and 220 rpm.
  5. On Day 3, 100 uL 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
    Sample Abs600 Reading Volume of Preloading Culture Volume of Preloading Media
    Positive control (1) 1.011 0.205 9.795
    Positive control (2) 2.261 0.0900 9.910
    Negative control 1.3 0.158 9.841
    Negative control (2) 0.734 0.287 9.713
    Device 1 (1) 0.443 1.493 8.507
    Device 1 (2) 0.138 1.980 8.020
    Device 2 (1) 0.425 2.515 7.485
    Device 2 (2) 0.136 2.020 7.980
    Device 3 (1) 0.507 0.426 9.574
    Device 3 (2) 0.543 0.395 9.604
    Device 4 (1) 0.306 0.743 9.257
    Device 4 (2) 0.409 0.538 9.462
    Device 5 (1) 0.195 0.266 9.734
    Device 5 (2) 0.494 0.438 9.562
    Device 6 (1) 1.597 0.357 9.643
    Device 6 (2) 1.58 0.130 9.870
    Media+chl 0.037
  6. Each culture was diluted to a target OD of 0.02 following the preloading culture and media volumes calculated by iGEM's Dilution Calculation Excel sheet in 12 mL of LB media + chloramphenicol in 50 mL falcon tubes wrapped in foil

Results