Part:BBa_K1692032

amilCP blue chromoprotein with RBS and promoter

This plasmid contains the amilCP blue chromoprotein gene found in part BBa_K592009 and the RBS and promoter from part BBa_K608002. For more information about this gene, please refer to the BBa_K592009 part page.

Usage and Biology

The chromoprotein amilCP is part of a family of GFP-like fluorescent proteins derived from reef-building corals of the class Anthozoa (Alieva et al., 2008). Similar to the GFP family of proteins, the β-barrel of amilCP encloses a chromophore (Tafoya-Ramírez et al., 2018). Along with other coral chromoproteins, amilCP is non-fluorescent and forms a tetramer, resulting in a fairly stable protein structure (Alieva et al., 2008; Tafoya-Ramírez et al., 2018). These GFP-like fluorescent proteins give corals their vivid and varied colors (Alieva et al., 2008).

Isolated from the species Acropora millepora, amilCP was first described in 2008, and is characterized by a strong color due to its very high molar extinction coefficient of 87 600 (Alieva et al., 2008). Its maximum excitation wavelength is 588nm (Alieva et al., 2008). Interestingly, amilCP’s max absorption is shifted into the red spectrum by ~10nm (592nm), hence appearing more blue than purple to the naked eye (Alieva et al., 2008). Such blue color is due to 2 mutations resulting in amino acid substitutions (Alieva et al., 2008).

Experiments

Protein Purification

Purified samples of protein allow further experiments to characterize their biological functions and interactions. Because this protein is not tagged with a short peptide sequence for affinity chromatography, the easiest method for purification of this protein may be by size-exclusion chromatography. Here, we propose a method for purification of amilCP via size-exclusion chromatography.

Buffer Preparation

For 40ml of a 1M phosphate buffer combine:

• 2.596g KH₂PO₄
• 3.644g K₂HPO₄
• Top off with distilled water to 40ml

For a 50mM (0.05M) phosphate buffer (makes 40ml):

• 2ml of 1M phosphate buffer
• Top off with distilled water to 40ml

References

Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M. E., Beltran-Ramirez, V., … Matz, M. V. (2008). Diversity and evolution of coral fluorescent proteins. PLoS ONE, 3(7). https://doi.org/10.1371/journal.pone.0002680

Tafoya-Ramírez, M. D., Padilla-Vaca, F., Ramírez-Saldaña, A. P., Mora-Garduño, J. D., Rangel-Serrano, Á., Vargas-Maya, N. I., … Franco, B. (2018). Replacing Standard Reporters from Molecular Cloning Plasmids with Chromoproteins for Positive Clone Selection. Molecules (Basel, Switzerland), 23(6). https://doi.org/10.3390/molecules23061328

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]