Difference between revisions of "Part:BBa K4488017"

Latest revision as of 13:32, 12 October 2022

fuGFPb

freeuse Green Fluorescent Protein-B, a blue-shifted GFP that fluoresces under blue light.

This part is an improvement of fuGFP (Part:BBa K3814004) which was originally designed to create a GFP variant not protected by patents and able to facilitate any kind of research.

Issue with fuGFP. The original fuGFP was optimised for green fluorescence detectable under UV by eye, however it absorbs less blue light than sfGFP. As a result, fuGFP has a primary excitation peak at ~400 nm and a less efficient secondary peak at ~470 nm. The decreased absorption at 470 nm is an issue for fuGFP because plate readers and other measuring devices usually have preset optical filters optimised for detecting the fluorescence of standard GFPs (sfGFP, eGFP) in the 470 nm range (Scordato & Schwartz, 2022). The signal of fuGFP at 470 nm is much weaker in comparison, and its primary excitation peak at 400 nm is susceptible to higher levels of background noise especially when working with media like LB which contain many molecules which fluoresce in this range (Billinton & Knight, 2001).

Improvement on fuGFP. Using PCR with degenerate primers we introduced the mutation in the fluorophore to create a diverse range of fuGFP variants including fuGFPb which contains the S66T mutation (figure 1). The improved part has a shifted excitation peak (485 nm) (figure 2) and absorbs more blue light in comparison to fuGFP, making it more similar to sfGFP instead. As a result, fuGFPb has the advantage of being more easily measured by plate readers that do not extend into the UV excitation range and can be used more efficiently with common optical filter sets. It has the potential as a fluorescent tag and is a suitable replacement for experiments requiring non-patented GFP with a similar fluorescence profile to standard GFPs especially if background fluorescence is an issue. Additionally, we have created a fusion protein of fuGFPb with a cellulose-binding domain allowing it to be immobilised on cellulose (Part:BBa K4488020).

References

Billinton, N & Knight, AW, 2001,, ‘Seeing the Wood through the Trees: A Review of Techniques for Distinguishing Green Fluorescent Protein from Endogenous Autofluorescence’, Analytical Biochemistry, vol. 291, no. 2, pp. 175–197, doi: 10.1006/abio.2000.5006.

Scordato, A & Schwartz, S (2022)., Fluorescence Filter Combinations, Nikon’s MicroscopyU, viewed October 2022, <https://www.microscopyu.com/techniques/fluorescence/nikon-fluorescence-filter-sets>.

Usage and Biology

Figure 1. fuGFP variants in pUS252, transformed into TOP10 E. coli. Plate shows the four different variant colours of fuGFP

Figure 2. Excitation/Emission Spectra for the Variants of fuGFPs. Measurement settings for the plate reader can be found here. Fluorescence intensity has been normalised so that all values are between 0 and 1 to reduce the measurement and sample artefacts. Four distinct fuGFPs are visualised. The original fuGFP is shown in purple, and is excited in the long wave UV range. The other three, fuGFPy, fuGFPb and fuGFPa, are all excited in the visible spectrum. fuGFPb (green lines on the plot) has a maximum excitation wavelength of 485nm. fuGFPy (yellow lines on the plot) has a maximum excitation wavelength of 488nm. fuGFPa (fuGFP-alanine, blue lines on the plot) has a maximum excitation wavelength of 482nm.

Sequence and Features