Fluorescence assays are amongst the most important measurement tools in biological experiments. However, the direct comparison of fluorescence data is problematic as data is often reported in different units or processed in different ways. This inability to directly compare results makes it harder to work collaboratively between laboratories and thus hinders the advancement of research. Therefore, it is important that a standardised protocol be established, so that groups around the world can achieve better, more streamlined fluorescence measurements. The Fourth Interlaboratory Measurement study aims to test the effectiveness of this standardised protocol by answering the important question: How close can the numbers be when fluorescence is measured all around the world using the same exact protocol?

Manchester iGEM 2017 is proud to have taken part in the interlab study for the first time. We hope our participation will help benefit other iGEM teams and the synthetic biology community as a whole.

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Protocol

Aseptic technique was maintained at all times.

Transformation

The E.coli strain used was Dh5α. The DNA provided in the distribution kit for the transformations was re-suspended using 10μl of dH2O. Competent cells were transformed using the following 8 devices, inside the plasmid backbone pSB1C3:

BBa_R0040 (Negative Control)
BBa_I20270 (Positive Control)
BBa_J364000 (Device 1)
BBa_J364001 (Device 2)
BBa_J364002 (Device 3)
BBa_J364003 (Device 4)
BBa_J364004 (Device 5)
BBa_J364005 (Device 6)

Transformations were done using our refined protocol (see additional work) and the transformed cells were subsequently plated on 8 LB agar plates containing chloramphenicol at a concentration of 25mg/ml. The plates were incubated overnight at 37°C.

Overnight Cultures

5ml of sterile LB broth was transferred to a total of 16 x 50ml falcon tubes, chloramphenicol was then added to each tube at a concentration of 25mg/ml. Two colonies were picked from each plate, each individual colony was transferred to a separate falcon tube. The tubes were incubated overnight at 37°C and 180 rpm.

Plate Reader Settings

Measurements of OD600 and fluorescence were taken using a BMG LabTech Clariostar plate reader, provided to us by the Manchester Institute of Biotechnology (MIB). OD600 measurements were all taken at 24°C with 35 flashes, and an orbital averaging of 3. Fluorescence measurements were taken with 8 flashes and a gain of 748. Excitation and emission wavelengths were 515-20 nm and 470-15 nm respectively. Pathlength correction was turned off for both measurements.

OD600 Reference Point

LUDOX S-40 was used as a single reference point to calibrate the plate reader. A ratiometric conversion factor was obtained to ensure that all Abs600 measurements were converted to OD600 measurements, whilst also taking into account differences in instruments.

100 µl of LUDOX was transferred into wells A1-D1 of a black, clear-bottom 96-well plate. 100 µl of distilled H2O was then added into wells A2-D2. Absorbance of LUDOX and H20 were then measured at 600nm. A ratiometric conversion factor of roughly 3.269 was obtained.

Fluorescence Standard Curve

The fluorescein stock was spun down for 30 seconds at 3000rpm and then re-suspended in 1mL of 1xPBS to produce a 2xstock solution (100μM). This was then further diluted with 1xPBS to a 1x stock solution with a final concentration of 50μM.

200µl of the fluorescein stock was transferred into wells A1-D1. The fluorescein stock was then serially diluted in four replicates (A2-A12, B2-B12, C2-C12, D2-D12) by mixing with 1x PBS to obtain 100µl of 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.048828125, and 0 µM of fluorescein solution. Fluorescence was then measured using a BMG LabTech ClarioStar plate reader, and a fluorescence standard curve generated (Figure 1). This standard curve was used to correct cell based readings to an equivalent fluorescein concentration and measure the concentration of GFP.

Cell Measurements

Overnight cultures of each device were diluted to a target OD of 0.02 and incubated at 37°C and 220 rpm, falcon tubes were wrapped in tin foil throughout. 500 µL of each device was transferred to an Eppendorf tube and put on ice at time points 0h, 2h, 4h, and 6h. 100μl of each sample was then transferred to 96 well plate using the provided set-up guidelines and measurements. OD and fluorescence measurements were taken simultaneously.

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Results

Figure 1: Standard curve of fluorescein fluorescence generated via 10-fold serial dilution using 4 standard replicates.

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Improvements

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Team:Manchester/InterLab

Interlab

Overview

Protocol

Transformation

Overnight Cultures

Plate Reader Settings

OD600 Reference Point

Fluorescence Standard Curve

Cell Measurements

Results

Improvements