Team:Macquarie Australia/InterLab

Background

Science requires the ability to perform and record reliable and repeatable measurements, some of the key aspects underpinning the scientific method. Reliability and repeatability provide an element of consistency and precision, respectively, as they rule out or take into account any anomalies or outliers that may occur. The development of technical standards including protocols and data-sheets,are helpful in achieving such reliability and repeatability and greatly increases the efficiency and predictability of synthetic biology laboratory work (Arkin, 2008).

Green fluorescent protein (GFP) is a molecular marker used in synthetic biology to report gene expression and successful transformation, and in fluorescence confocal microscopy to label or characterise proteins and pathways. Equipment used to measure its expression is usually present in most laboratories, making it an accessible marker to use. Fluorescein, a bright green fluorophore, and its derivatives for example Fluorescein Isothiocyanate (FITC), have high absorptivity, excellent fluorescence and good water solubility, characteristics making them the most common fluorescent reagents for biological research (ThermoFisher Scientific). Together, these markers can be used as powerful tools for synthetic biology measurement and analysis, especially when used in technical standards. Without such standards, it can be difficult to obtain reliable and repeatable results, as cells are inherently complex biological systems that give differing results depending on the methods and machinery used. Absolute standard measures are required to prevent results from straying away from that which is expected (Torrance and Kahl, 2014).

Aims

This year H₂ydroGEM (Macquarie University Australia) participated in the 4th International InterLab Measurement Study. The aim of this study was to follow the detailed protocols provided by iGEM to obtain fluorescence measurements and fill in data analysis forms which could then be used by the iGEM Measurement Committee to directly make comparisons to those provided by other teams. The objective of them doing so was to create a standardised protocol that could be used to measure GFP fluorescence and OD₆₀₀ absorbance on any plate reader.

The goal was to measure absorbances and GFP fluorescence of E.coli transformants in common units. These transformants came from six test devices, each with varied constitutive promoters, ribosome binding sites and design elements.

Materials and Methods

Calibration

The 600 nm absorbances of 100% LUDOX and H₂O replicates in a well plate were measured and recorded to calculate the OD₆₀₀ reference point.
Fluorescein Measurement

Serial dilutions were then performed for four replicates.
Their fluorescence was measured using a BMG Labtech Fluostar Optima plate reader plate reader to construct a standard fluorescence curve.

Cell Measurement

K-12 E. coli DH5α cells were transformed with the supplied plasmids (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) and grown on chloramphenicol supplemented agar plates.
Two colonies from each plate were chosen and grown up in liquid culture overnight. From each of the 2 liquid cultures, 4 replicates were made.
At each time point; 0 hours, 2 hours, 4 hours, 6 hours, a sample was taken from the replicate culture and placed on ice. These samples were then pipetted into a well plate for the respective time point, wrapped in foil and placed in the fridge until measurements were taken.
GFP fluorescence and sample absorbances (Abs₆₀₀) for all replicates in each time point well plate were measured using BMG Labtech Fluostar Optima plate reader.

Click here for full protocol

Results and Discussion

Colony GFP fluorescence results - As seen in Fig. 1., there was a sharp increase in average fluorescence in both colonies of Test Device 2 and Test Device 4, during the last 2 hours of incubation. This was similar to the positive controls. Test Device 2 - Colony 1 had the greatest average fluorescence out of all the Test Devices Positive (contained constitutively expressed promoter) and negative (containing TetR constitutively repressed promoter) controls were measured so we could make comparisons with results from the test devices. As expected, the negative controls had the lowest readings, closely followed by Test Device 3 - Colony 2 and Test Device 1 - Colony 2.

Colony absorbance results - Despite Test Device 3 - Colony 2 not displaying significant fluorescence, absorbance level was high indicating growth. Most replicates reached peak absorbance at 4 hours. The greatest variance between replicates was in Test Device 6 - Colony 2, as evident through the largest standard deviation range The standard error bars for the averages of the replicates of both GFP fluorescence and colony absorbance were similarly not very large.

Calibration using fluorescein - The fairly linear graph of FITC standard curve (Fig 3) obtained from the serial dilutions, indicated increased fluorescence with increased fluorescein concentration. The mean value of the replicates was reliable as the standard deviation or standard error was very small at each concentration.

Conclusions

It is hoped the measurements provided by our team are consistent with other teams and can therefore be used in the establishment of a GFP measurement protocol that can be used with any plate reader. We look forward to participating in future Interlab projects.

References

Arkin, A. (2008). Setting the standard in synthetic biology. Nature biotechnology, 26(7), pp. 771-4.

ThermoFisher Scientific. Fluorescein (FITC) [Online]. Available: https://www.thermofisher.com/au/en/home/life-science/cell-analysis/fluorophores/fluorescein.html [Accessed 11 October 2017].

Torrance, A. W. and Kahl, L. J. (2014). Bringing standards to life: synthetic biology standards and intellectual property. Santa Clara High Technology Law Journal. 30, pp. 199-230.