Overview

The Interlab Study, developed by the Measurement Committee of iGEM as a way to standardize measurements for green fluorescent protein (GFP), tackles the foundational synthetic biology problem of a lack of reliable measurements for experiments. With an international standard set down, scientists can go beyond utilizing relativity comparisons for results, to conduct quantitative reproducibility studies across labs. This Interlab experiment, carried out by McMaster iGEM with plate readers, aims to produce comparable GFP measurements as a contribution for the international standardization of GFP values. The parts we used were BBA_J364000 (1), BBa_J364001 (2), BBa_J364002 (3), BBa_J36003 (4), BBa_J364004 (5), BBa_J364005 (6), and positive control BBa_120270 with negative control BBa_R0040. All parts were located in pSB1C3 plasmid and were modified for chloramphenicol resistance.

Methods and Materials

Plate Reader

Plate Reader: TECAN Infinite M1000 Plate: Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)
Abs OD600

Wavelengths: 600 nm
Number of Flashes: 20
Settle Time: 0 ms
Temperature: 26.1 degrees Celsius

Fluorescence

Excitation:485 nm
Emission: 535 nm
Excitation Bandwidth: 5 nm
Emission Bandwidth: 5 nm
Optics: Bottom Reading
Gain: Optimal (automatic)
Number of Flashes: 50
Flash Frequency: 400 Hz
Integration Time: 20 us
Lag Time: 0 us
Settle Time: 0 ms
Part of Plate: A1-H10
Temperature: 26.8 degrees Celsius

OD600 Reference Point

Materials

1 mL LUDOX (provided for by iGEM Headquarters)
Distilled H2O
96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)

Methods

Add 100 uL of LUDOX into A1, B1, C1, and D1 wells
Add 1 mL LUDOX into a separate cuvette
Add 100 uL of dH2O into wells A2, B2, C2, D2 wells
Add 1 mL dH2O into a separate cuvette
Image using plate reader (TECAN Infinite M1000)

Protocol of Fluorescein Fluorescence Standard Curve

Materials:

Fluorescein
10 mL 1x PBS (phosphate buffered saline)
96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)

Methods:

Spin down fluorescence stock tube until pellet is at bottom of tube
Resuspend 100 uM stock Fluorescein in 1 mL of 1x PBS to obtain 2x fluorescein stock solution.
Ensure that Fluorescein is properly dissolved.
Dilute 2x fluorescin stock solution with 1x PBS to make a 1x fluorescein solution. Resulting concentration of fluorescein stock solution should be 50 uM (otherwise known as 500 uL of 2x fluoresceine in 500 uL of 1x PBS that makes 1 mL of 50 uM of 1x fluorescein solution).
Prepare for serial dilutions across columns 1 to 11. Ensure column 12 has PBS buffer only.
- Pipette 200 uL of 1x fluorescein stock into wells A1, B1, C1, D1.
- Pipette 100 uL of PBS in wells A2, B2, C2, D2 ... A12, B12.
- Transfer 100 uL of fluorescein stock solution from A1 to A2.
- Mix A2 by pipetting up and down 3x and transfer 100 uL into A3.
- Mix A3 by pipetting up and down 3x and transfer 100 uL into A4.
- Repeat 5c to 5e for A4, A5, A6.... to A11.
- Mix A11 by pipetting up and down 3x and then transfer 100 uL into liquid waste. Ensure that A12 is PBS only
Repeat step 5 for columns B, C, and D.
Measure fluorescence of all samples in the plate reader (settings listed above in Plate Reader Setup menu).
Record data in Fluorescein Standard Curve excel sheet and graph.

Cell Measurements

Materials:

Competent Cells (Escherichia coli strain DH5 alpha)
Luria Bertani media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 ug/mL)
50 mL Falcon tube (covered in foil to block light)
Incubator at 37 degree Celsius
1.5 mL eppendorf tubes for sample storage
Ice bucket with ice
Pipettes
96 well plate, black with transparent bottom (Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)

Plasmids

BBA_J364000 (1), J23101+I13504
BBa_J364001 (2), J23106+I13504
BBa_J364002 (3), J23107+I13504
BBa_J36003 (4), J23101.BCD2.E0040.B0015
BBa_J364004 (5), J23106.BCD2.E0040.B0015
BBa_J364005 (6), J23107.BCD2.E0040.B0015
Positive control BBa_120270
Negative control BBa_R0040

Methods:

Transform Esherichia coli DH5 alpha with following the following plasmids

Device 1: BBA_J364000 (1), J23101+I13504
Device 2: BBa_J364001 (2), J23106+I13504
Device 3: BBa_J364002 (3), J23107+I13504
Device 4: BBa_J36003 (4), J23101.BCD2.E0040.B0015
Device 5: BBa_J364004 (5), J23106.BCD2.E0040.B0015
Device 6: BBa_J364005 (6), J23107.BCD2.E0040.B0015
Positive control BBa_120270
Negative control BBa_R0040.

Pick 2 colonies from each plate and inoculate on a 5-10 mL LB medium + Chloramphenicol.

18 tubes of 5 mL LB with 5 uL of chloramphenicol (25 ug/mL)

Grow cells overnight for 16-18 hours at 37 degrees Celsius and at 220 rpm.
Measure cell growth with spectrophotometer:

Set spectrophotometer to read OD600.
Measure OD600 of overnight cultures (placed in 1 mL spectrophotometer cuvettes) after calibrating instrument with distilled H2O.
Record data and transfer data into the Dilution Calculation Sheet_1 provided by iGEM Headquarters.
Dilute all cultures to a target OD600 of 0.02 in 12 mL LB medium + Chloramphenicol in a 50 mL capacity Falcon Tube covered with aluminum foil to block light.
Incubate cultures at 37 degrees Celsius and at 220 rpm.
Take 500 uL samples of each of the cultures at 0 hours, 2 hours, 4 hours and 6 hours of incubation.
Total samples taken should be 16 samples, with 2 samples from each 8 devices at each time point.
Do not reuse samples

Place samples on ice in meantime while pipetting them into the plate to be measured.

When pipetting into the clear bottom plate to be measured by the TECAN Infinite M1000 plate reader using absorbance and fluorescence:

Pipette both samples into replicates of 4 each so that there are 4 wells, each filled with 100 uL, for each sample.
This totals to 8 wells of 100 uL per device.