Difference between revisions of "Part:BBa K3814004"

Latest revision as of 10:40, 12 October 2022

free-use GFP (fuGFP)

fuGFP, short for ‘free-use GFP’, is an open source GFP developed by Mark Somerville in the Coleman lab at the University of Sydney. The rationale for developing this GFP variant was to make a superfolding GFP that was not protected by patents (unlike the original sfGFP), thus facilitating all kinds of synbio research, whether or not this has commercial intent. The mechanism for moving the GFP off-patent was to use DNA shuffling to derive a synthetic gene encoding a protein with <80% amino acid identity to sfGFP, since the patent claims everything at >80% identity. fuGFP preferentially absorbs long-wave UV light and emits green. Note that this is different to sfGFP, which has an S65T mutation that makes it absorb more blue light. fuGFP is available as pUS252 in AddGene.

Figure 1. Expression of fuGFP in E.coli strain TOP10.

References

Coleman, N., & Somerville, M. (2019, May). The Story of Free Use GFP (fuGFP). Small Things Considered. https://schaechter.asmblog.org/schaechter/2019/05/the-story-of-free-use-gfp-fugfp.html

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]

2022 USYD Sydney Australia's Contribution

Authors: Oliver Nicholls and Donovan Wu

Introduction

Throughout our project we have used fuGFP as a fluorescent marker. We have characterised the absorbance spectrum of fuGFP and compared it to sfGFP. Furthermore, we were able to calculate the extinction coefficient of fuGFP through our experiments.

Results

Figure 1. Theoretical absorption spectra of fuGFP and sfGFP at 1 mg/ml between 335-550 nm.

Our team has contributed to the characterisation of this part by calculating a theoretical absorbance spectrum 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.

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).

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

Figure 2. Excitation (340-520nm) and emission (480-560nm) spectra of fuGFP and sfGFP.

Table 1. Excitation peak, emission peaks and extinction coefficient of fuGFP
Protein	fuGFP
Excitation peak (nm)	395
Emission peak (nm)	503
Extinction coefficient at 395nm (M⁻¹ cm⁻¹)	27618

References

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).

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.

@@ Line 31: / Line 31: @@
 ===<h1>2022 USYD Sydney Australia's Contribution</h1>===
+<b>Authors</b>: Oliver Nicholls and Donovan Wu
 <h2> Introduction </h2>
@@ Line 40: / Line 42: @@
 Our team has contributed to the characterisation of this part by calculating a theoretical absorbance spectrum 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.
-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.
+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).
+Excitation and emission spectra of 200µL from the same dilutions and stock solutions were measured using the plate reader. Excitation values were measured from 340-520nm and emission values were measured from 480-560nm for both sfGFP and fuGFP and normalised against their peak RFU values (Figure 2).
+[[File:fuGFP and sfGFP spectra.png|centre|thumb|700px| Figure 2. Excitation (340-520nm) and emission (480-560nm) spectra of fuGFP and sfGFP.]]
-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.
+{| class="wikitable" style="margin:auto"
+|+ Table 1. Excitation peak, emission peaks and extinction coefficient of fuGFP
+|-
+! Protein !! fuGFP
+|-
+| Excitation peak (nm) || 395
+|-
+| Emission peak (nm) || 503
+|-
+| Extinction coefficient at 395nm (M<sup>−1</sup> cm<sup>−1</sup>) || 27618
+|}
 <h2>References</h2>