amajLime, yellow-green chromoprotein (incl RBS & J23110)
This chromoprotein from the coral Anemonia majano, amajLime (also known as amajCFP or amFP486), naturally exhibits strong color when expressed. The protein has an absorption maximum at 458 nm giving it a yellow-green 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 amajLime has significant sequence homologies with proteins in the GFP family.
Team: Humboldt_Berlin 2019
ExPASy ProtParam Results:
Number of amino acids: 228
Molecular weight: 25185.49 Da
Theoretical pI: 7.27
Total number of negatively charged residues (Asp + Glu): 22
Total number of positively charged residues (Arg + Lys): 22
Ext. coefficient 26150 M-1 cm-1
Abs 0.1% (=1 g/l) 1.038, assuming all pairs of Cys residues form cystines
Ext. coefficient 25900 M-1 cm-1
Abs 0.1% (=1 g/l) 1.028, assuming all Cys residues are reduced
Instability index: The instability index (II) is computed to be 27.07. This classifies the protein as stable.
Aliphatic index: 51.71
Grand average of hydropathicity (GRAVY): -0.379
In order to measure absorbance and fluorescence spectra of amajLime we transformed the construct (consisting of BBa_J23110 Promotor, BBa_B0034 RBS and amajLime coding sequence) into E. coli (DH10B Competent Cells). After cultivation and we lysed the harvested cells according to this protocol.
The fluorescence and absorbance spectra were 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 with a peak at 454 nm (compared to an excitation maximum of 458 nm in the literature )including the respective standard deviation. Figure 2 shows the fluorescence spectrum with a peak at 493 nm (compared to an excitation maximum of 486 nm in the literature ) including the respective standard deviation. In figure 3 the joined relative absorbance and fluorescence spectra are shown.
|Table 1. Parameters utilized for absorbance and fluorescence spectra|
|Number of Samples||24|
|Wavelength Step Size||2|
|Absorbance Scan: Excitation Wavelength Measurement Range (nm)||[300-800]|
|Fluorescence Scan: Emission Wavelength Measurement Range (nm)||[475-550]|
|Fluorescence Scan: Excitation Wavelength (nm)||445|
|Number of Flashes||25|
|Integration Time (µs)||20|
|Lag Time (µs)||0|
|Settle Time (ms)||0|
 Matz, Mikhail V., et al. "Fluorescent proteins from nonbioluminescent Anthozoa species." Nature biotechnology 17.10 (1999): 969-973.
 Henderson, J. Nathan, and S. James Remington. "Crystal structures and mutational analysis of amFP486, a cyan fluorescent protein from Anemonia majano." Proceedings of the National Academy of Sciences of the United States of America 102.36 (2005): 12712-12717.
 Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.
 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
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC