Team:TP-CC San Diego/InterLab

Interlab

Click Here for the Interlab Excel

Introduction

This year’s interlab study is intended to answer the main question of the relativity of fluorescence when measured at different parts of the world. To make this data more reliable, iGem has asked to test some RBS devices to make gene expression reliable and precise. For this year’s interlab study, TP-CC San Diego has decided to do the plate reader protocol along with transforming the plasmids from Kit 7.

Method

OD​600​ Reference point

Procedure:

  1. Add 100 µl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
  2. Add 100 µl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
  3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
  4. Record the data in the table below or in your notebook
  5. Import data into Excel (OD600 reference point tab)​ Sheet_1 provided

Prepare the fluorescein stock solution:

Procedure:

  1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube
  2. Prepare 2x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS. [Note​: it is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip – if any particulates are visible in the pipette tip continue to mix the solution until they disappear.]
  3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 µM (500µL of 2x fluorescein in 500 µL 1x PBS will make 1 mL of 50 µM (1x) fluorescein solution.)

OD​600​ Reference point

Procedure:

  1. Add 100 µl of PBS​ into wells A2, B2, C2, D2....A12, B12, C12, D12
  2. Add 200 µl​ of fluorescein 1x stock​ solution into A1, B1, C1, D1
  3. Transfer 100 µl of fluorescein stock solution from A1 into A2.
  4. Mix A2 by pipetting up and down 3x and transfer 100 µl into A3…
  5. Mix A3 by pipetting up and down 3x and transfer 100 µl into A4...
  6. Mix A4 by pipetting up and down 3x and transfer 100 µl into A5...
  7. Mix A5 by pipetting up and down 3x and transfer 100 µl into A6...
  8. Mix A6 by pipetting up and down 3x and transfer 100 µl into A7...
  9. Mix A7 by pipetting up and down 3x and transfer 100 µl into A8...
  10. Mix A8 by pipetting up and down 3x and transfer 100 µl into A9...
  11. Mix A9 by pipetting up and down 3x and transfer 100 µl into A10...
  12. Mix A10 by pipetting up and down 3x and transfer 100 µl into A11...
  13. Mix A11 by pipetting up and down 3x and transfer 100 µl into liquid waste
  14. Repeat dilution series for rows B, C, D
  15. Measure fluorescence of all samples in all standard measurement modes in instrument
  16. Record the data in your notebook
  17. Import data into Excel (fluorescein standard curve tab)​ Sheet_1 provided

Cell measurement protocol

Procedure:

  1. Positive control
  2. Negative control
  3. Test Device 1: J23101+I13504
  4. Test Device 2: J23106+I13504
  5. Test Device 3: J23117+I13504
  6. Test Device 4: J23101.BCD2.E0040.B0015
  7. Test Device 5: J23106.BCD2.E0040.B0015
  8. Test Device 6: J23117.BCD2.E0040.B0015
  9. : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
  10. Set your instrument to read OD600 (as OD calibration setting)
  11. Measure OD600 of the overnight cultures
  12. Record data in your notebook
  13. Import data into Excel (Dilution Calculation​) Sheet_1 provided
  14. Dilute the cultures to a target OD600 of 0.02 (see the volume of preloading culture and media in Excel (Dilution Calculation​) Sheet_1) in 12 m​l LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
  15. Incubate the cultures at 37°C and 220 rpm.
  16. Take 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation. (At each time point, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 samples per time point)
  17. Place samples on ice.
  18. At the end of sampling point you need to measure your samples (OD and Fl measurement), see the below for details.
  19. Record data in your notebook
  20. Import data into Excel (cell measurement tab​) Sheet_1 provided

Data

uM Fluorescein

Fluorescein Standard Curve

Fluorescein Standard Curve (log scale)

uM Fluorescein/a.u.

Raw Plate Readings

Fluorescence Raw Readings:

Abs600 Raw Readings:

Results

Raw Abs600

Raw Fluorescence

OD - Background

Fluorescence - Background

uM Fluorescein / OD600

Summary Statistics