Part:BBa_K864100

Super Yellow Fluorescent Protein 2 (SYFP2)

This part codes for the bright yellow fluorescent protein SYFP2. SYFP2 is a GFP based monomeric protein with optimized folding, maturation and a narrow fluorescence emission spectrum with a maximum of 515 nm. Bacteria expressing SYFP2 are reported to be 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus (Kremer et al 2006). Mutations compared to wtGFP amino acid sequence (GenBank Accession number M62653) are F46L F64L S65G S72A M153T V163A S175G T203Y A206K. See the article listed in references. The sequence has been codon optimized for expression in E coli by DNA 2.0.

Kremers et al reported the following data:

Excitation peak: 515 nm. Emission peak: 527 nm. Relative brightness to EYFP2(Q96K) in E coli: 12.0. Extinction Coefficient (M-1 cm-1): 101000. Quantum Yield: 0.68.

Usage and Biology

This part is useful as a reporter.

iGEM12_Uppsala_University. Fluorescence on UV table. The images above show E coli constitutively expressing SYFP BBa_K864100 using promoters with different strengths.

Team Warsaw 2013 designed sfYFP and compared it to SYFP2. Measurment was conducted on pSB1A3 plasmid with J23100 promoter and B0034 RBS in living cells in RF.

Fig 1. – Fluorescence of sfYFP and SYFP2 compared to sfGFP (BBa_I746908).

-	Samples			Arithmetic mean	Standard deviation
sfGFP	100,60	100,37	99,02	100,00	0,85
sfYFP	218,87	216,51	217,87	217,75	1,19
SYFP2	424,70	418,26	412,40	418,45	6,15

Tab 1. – Fluorescence of sfYFP and SYFP2 compared to sfGFP (BBa_I746908).

R9-SYFP2-H6 Improvement by iGEM UCopenhagen 2017

An improved version of the SYFP2 reporter with an N-terminal Cell-Penetrating peptide (CPP) signal was constructed by the UCopenhagen 2017 iGEM Team. The addition of the nona-argigine CPP tag was shown to give the SYFP2 biobrick the ability stain both E. coli and P. putida cells, presumably by causing internalisation. In addition the improved biobrick carries a C-terminal hexa-histidine tag. For more information please visit the improved part CPP-SYFP2.

Figure 3: Fluorescence of E. coli cells treated with fluorescent protein. Cells were treated with either purified mTag BFP (A-B), CPP-mTag BFP (C-D), SYFP2 (E-F), or CPP-SYFP2 (G-H) for 10 min and before being washed twice. Cells treated with CPP-tagged protein emitted noticeably more fluorescence.

Figure 4: Fluorescence of P. putida cells treated with fluorescent protein. Cells were treated with either purified mTag BFP (A-B), CPP-mTag BFP (C-D), SYFP2 (E-F), or CPP-SYFP2 (G-H) for 10 min before being washed twice. Cells treated with CPP-tagged protein emitted noticeably more fluorescence.

Author: Jon Fugl

References

[http://pubs.acs.org/doi/abs/10.1021/bi0516273] Kremers, G.-J., Goedhart, J., van Munster, E. B., & Gadella, T. W. J. (2006). Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry, 45(21), 6570–6580. doi:10.1021/bi0516273

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]