Part:BBa_K1033917
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 (300-1000 nm) was performed to what iGEM Upsala 2013 has done, which is 300-800 nm. Results can be seen in Figures 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, 100 uL of each chromoprotein sample was mixed with 100 uL of different pH-samples.
Figure 1: Absorbance spectrum of saline.
Figure 2: Full spectrum analysis of gfasPurple.
Figure 3: Duplicate of the full spectrum analysis of gfasPurple.
Figure 4: Full spectrum pH-analysis of gfasPurple.
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.
Liquid culture of E. coli expressing gfasPurple
Cell pellet after centrifugation
Cleared cell lysate
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 | ||
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
[http://www.ncbi.nlm.nih.gov/pubmed/18648549] Alieva, Naila O., et al. "Diversity and evolution of coral fluorescent proteins." PLoS One 3.7 (2008): e2680.
[2] 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |