Reporter

Part:BBa_K1033917

Designed by: Sabri Jamal   Group: iGEM13_Uppsala   (2013-09-17)

gfasPurple, purple chromoprotein (incl RBS, J23110)

This chromoprotein from the coral Galaxea fascicularis, gfasPurple (also known as gfasCP), naturally exhibits strong color when expressed. The protein has an absorption maximum at 577 nm giving it a purple color visible to the naked eye. Compared to many other chromoproteins, such as amilCP (BBa_K592009), amilGFP (BBa_K592010), spisPink (BBa_K1033932), asPink (BBa_K1033933) and aeBlue (BBa_K864401), the color development is slower. The color is readily observed in both LB or on agar plates after 24-48 hours of incubation. The protein gfasPurple is closely related to amilCP (BBa_K592009) and has significant sequence homologies with proteins in the GFP family.

Characterisation

The iGEM team of Rotterdam 2019 characterized this part more as part of the bronze criteria. [1]An addition to the characterization tests is to see whether there will be a change in the absorbance spectrum when the proteins are suspended in acidic or basic environments.

First, full-spectrum analysis with a Synergy™ LX Multi-Mode Microplate Reader(300-1000 nm) was performed to what iGEM Upsala 2013 has done, which is 300-800 nm (Figure 1-3).

The second characterization experiment (figure 4 and 5) was to test the effect of pH: 0.3, 4.3, 7.3, 10.3 and 14. The tests have been performed twice in different wells of a 96-wells plate with a Synergy™ LX Multi-Mode Microplate Reader, 100 uL of each chromoprotein sample was mixed with 100 uL of different pH-samples. Data of the Microplate Reader: https://2019.igem.org/wiki/images/1/18/T--Rotterdam_HR--excelfilepH.xlsx 800px-T--Rotterdam_HR--result10.jpg

Figure 1: Absorbance spectrum of saline.

800px-T--Rotterdam_HR--result13.jpg

Figure 2: Full spectrum analysis of gfasPurple.

800px-T--Rotterdam_HR--result14.jpg

Figure 3: Duplicate of the full spectrum analysis of gfasPurple.

T--Rotterdam_HR--result18.jpg

Figure 4: Full spectrum pH-analysis of gfasPurple.

T--Rotterdam_HR--design2.png

Figure 5: Duplicate of the full spectrum pH-analysis of gfasPurple.


Characterisation

Team: Humboldt_Berlin 2019


ExPASy ProtParam Results:


Number of amino acids: 220

Molecular weight: 24816.17 Da

Theoretical pI: 7.61

Total number of negatively charged residues (Asp + Glu): 24

Total number of positively charged residues (Arg + Lys): 25


Extinction coefficients:

Ext. coefficient 37610 M-1 cm-1

Abs 0.1% (=1 g/l) 1.516, assuming all pairs of Cys residues form cystines

Ext. coefficient 37360 M-1 cm-1

Abs 0.1% (=1 g/l) 1.505, assuming all Cys residues are reduced

Instability index: The instability index (II) is computed to be 39.71. This classifies the protein as stable.

Aliphatic index: 61.50

Grand average of hydropathicity (GRAVY): -0.544

In order to measure the absorbance spectrum of gfasPurple we transformed the construct (consisting of BBa_J23110 Promotor, BBa_B0034 RBS and gfasPurple coding sequence) into competent E. coli. After cultivation and we lysed the harvested cells according to this protocol.


The absorbance spectrum was measured for 24 samples of 150 µl lysate in 96-well plate on TECAN Plate Reader Infinite 200 Pro. In figure 1 you can see the absorbance spectrum including the respective standard deviation with a peak at 580 nm (compared to an excitation maximum of 577 nm in the literature [2]).


Table 1. Parameters utilized for absorbance spectrum
Parameter Value
Number of Samples 24
Wavelength Step Size 2
Absorbance Scan: Excitation Wavelength Measurement Range (nm) [300-800]
Number of Flashes 25
Settle Time (ms) 0


Figure 1. gfasPurple absorbance spectrum

Source

Galaxea fascicularis. The protein was first extracted and characterized by Alieva et. al. under the name gfasCP (GenBank: DQ206394.1). This version is codon optimized for E coli by Genscript.

References

[2] Alieva, Naila O., et al. "Diversity and evolution of coral fluorescent proteins." PLoS One 3.7 (2008): e2680.

[3] Liljeruhm, Josefine et al. “Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.” Journal of biological engineering vol. 12:8. 10 May. 2018, doi:10.1186/s13036-018-0100-0


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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