Difference between revisions of "Part:BBa K4488017"

Revision as of 01:06, 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. The original fuGFP absorbs less blue light compared to sfGFP. The decreased absorption of blue light is an issue for fuGFP because plate readers with special UV filters are required to detect the excitation wavelength, restricting its accessibility. Thus, using PCR with degenerate primers we introduced the S66T mutation in the fluorophore to create fuGFPb. The improved variant has a blue-shifted excitation peak (485 nm) (figure 1) and absorbs more blue light making it more similar to sfGFP. As a result, fuGFPb has the advantage of being more easily measured as plate readers that do not extend into the UV range for excitation can be used. Furthermore, it has promising potential as a fluorescent tag. Additionally, we also created a fusion protein of fuGFPb with a cellulose-binding domain allowing it to be immobilised on cellulose (Part:BBa K4488020).

Usage and Biology

Figure 1. 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 green, and is excited in the long wave UV range. The other three, fuGFPy, fuGFPb and fuGFPa, are all excited in the visible spectrum. fuGFPb (blue 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, orange lines on the plot) has a maximum excitation wavelength of 482nm.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 151
1000
COMPATIBLE WITH RFC[1000]

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 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. The original fuGFP absorbs less blue light compared to sfGFP. The decreased absorption of blue light is an issue for fuGFP because plate readers with special UV filters are required to detect the excitation wavelength, restricting its accessibility. Thus, using PCR with degenerate primers we introduced the S66T mutation in the fluorophore to create fuGFPb. The improved variant has a blue-shifted excitation peak (485 nm) (figure 1) and absorbs more blue light making it more similar to sfGFP. As a result, fuGFPb has the advantage of being more easily measured under UV and has promising potential as a fluorescent tag. Additionally, we also created a fusion protein of fuGFPb with a cellulose-binding domain allowing it to be immobilised on cellulose (Part:BBa K4488020).
+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. The original fuGFP absorbs less blue light compared to sfGFP. The decreased absorption of blue light is an issue for fuGFP because plate readers with special UV filters are required to detect the excitation wavelength, restricting its accessibility. Thus, using PCR with degenerate primers we introduced the S66T mutation in the fluorophore to create fuGFPb. The improved variant has a blue-shifted excitation peak (485 nm) (figure 1) and absorbs more blue light making it more similar to sfGFP. As a result, fuGFPb has the advantage of being more easily measured as plate readers that do not extend into the UV range for excitation can be used. Furthermore, it has promising potential as a fluorescent tag. Additionally, we also created a fusion protein of fuGFPb with a cellulose-binding domain allowing it to be immobilised on cellulose (Part:BBa K4488020).
 ===Usage and Biology===