Part:BBa_K3814004

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]

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

File:FuGFP vs sfGFP Abs

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Our team has contributed to the characterisation of this part by comparing the absorbance spectra of fuGFP to sfGFP which makes it easier for future teams to use. We used purified fuGFP and sfGFP (1 mg/mL) and made 1:6 and 1:9 dilutions of both proteins. The absorbances of the diluted samples were measured in the plate reader 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.