Difference between revisions of "Part:BBa K1033917"

Revision as of 13:34, 14 October 2019

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

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 J23110 Promotor, RBS B0034 and gfasPurple coding sequence) into competent E. coli. After cultivation and we lysed the harvested cells according to this protocol.

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  Liquid culture of E. coli expressing gfasPurple

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  Cell pellet after centrifugation

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  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 relative 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]).

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

[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.

[1] 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