Contribution

Basic Part Characterisation - fuGFP

Free-use GFP (fuGFP) was designed by Mark Somerville at the University of Sydney with the aim of creating an unpatented GFP variant that would facilitate any kind of synthetic biology research by any organisation. The part (BBa_K3814004) was added to the iGEM parts registry by the 2021 University of Sydney Team.

Our team has contributed to the characterisation of this part by calculating a theoretical absorbance spectrum (Figure 1) for a 1 mg/ml concentration solution of fuGFP from experimental data, enabling future teams to use spectroscopy to quantify the concentration of this part.

Image of theoretical absorbance spectra for fuGFP and sfGFP. sfGFP shows an absorbance peak exceeding 1.5 in the range of approximately 475-500 nanometers, while fuGFP shows an absorbance peak at 1 in the wavelength range of 390-400 nanometers.
Figure 1. Theoretical absorption spectra of fuGFP and sfGFP at 1 mg/ml between 335-550 nm.

The absorbances were measured in the photospectrometer between 300–700 nm and the average was graphed (Figure 1). We found that fuGFP exhibits peak absorbance at 395 nm with a secondary peak at 475 nm. In comparison, sfGFP displays only a single peak at 490 nm.

We obtained purified fuGFP and sfGFP stock solutions from Mark Somerville and made 1:5 and 1:8 dilutions of both proteins. In addition to these concentrations, we made up a 1:9 dilution in 0.2 M NaOH. This deprotonates the chromophore of the fluoroproteins such that they take on a homologous conformation between sfGFP and fuGFP, resulting in identical absorption spectra at these wavelengths (Ward et al. 1981). The extinction coefficient of GFPs with this denatured chromophore at 446 nm is 44,000 M−1 cm−1 (Bomati et al. 2015).

The Beer-Lambert law was used to back calculate the concentrations of the original solutions, which were determined to be 1.5 mg/ml and 1.4 mg/ml for fuGFP and sfGFP respectively. From these concentrations, the extinction coefficient of fuGFP was calculated to be 27618 M−1 cm−1. To normalise the absorption values for a 1 mg/ml solution, we divided the absorption values by their respective concentrations and graphed the resulting spectra (Figure 1).

Image of Excitation (340 to 520 nanometers) and Emission (480 to 560 nanometers) spectra of fuGFP and sfGFP. it shows that while emission peaks are similar for the two GFP spectra, fuGFP excites with light of a lower wavelength (about 400 nm) than sfGFP (about 500 nm).
Figure 2. Excitation (340-520 nm) and Emission (480-560 nm) spectra of fuGFP and sfGFP.

Excitation and emission spectra of 200 µL from the same dilutions and stock solutions were measured using the plate reader. Excitation values were measured from 340-520 nm and emission values were measured from 480-560 nm for both sfGFP and fuGFP and normalised against their peak RFU values (Figure 2).

References

Ward, W., DeLuca, M. and McElroy, W., 1981. PROPERTIES OF THE COELENTERATE GREEN-FLUORESCENT PROTEINS - Bioluminescence and Chemiluminescence. 1st ed. Saint Louis: Elsevier Science, pp.235–242.

Bomati, E.K., Haley, J.E., Noel, J.P., and Deheyn, D.D., 2015. Spectral and structural comparison between bright and dim green fluorescent proteins in Amphioxus. Scientific Reports, 4(1).