Part:BBa_K5527001

amilCP purple, Purple chromoprotein

This part is the coding sequence of a purple chromoprotein. The part was made by a substitution at position 157, which in the original amilCP (BBa_K592009) is asparagine, to alanine.

Usage and Biology

amilCP is a chromoprotein from the coral Acropora millepora, which can give it a blue color. It belongs to a larger group of GFP-like proteins, but unlike GFP it isn’t fluorescent. It occurs as a homodimer, where each monomer where each monomer makes up a beta-barrel consisting of 11 beta-strands. The chromophore, made up of Gln62, Tyr63 and Gly64, is connected to an alpha-helix running through the barrel (Ahmed et al. 2022).

This part can be used as a reporter gene. In contrast to the common fluorescent reporters such as GFP, this part absorbs light of the visible spectra. This means no special equipment is needed to detect activity, and the cells don’t have to be exposed to UV light, which could harm them (Liljeruhm et al. 2018).

Characterization

Figure 1: Wild type amilCP, purple, light purple and yellow mutants in liquid culture.

Figure 2: Pellets of wild type amilCP, purple, light purple and yellow mutants.

Figure 3: Pictures of LB-agar plates with different amilCP mutants. Top left: purple mutant (this part). Top right: wild type amilCP. Bottom left: yellow mutant. Bottom right: light purple mutant.

Figure 4: Absorbance spectrum. Maximum at 585 nm.

Figure 5: Box plots of maturation halftimes for WT amilCP, Light purple and purple mutations.

The maturation halftime, defined as the time it takes the chromoprotein to reach half of its intensity, was measured and estimated using a camera and a computer program, opencv, to analyze the pictures.
The E.coli containing the different mutant amilCPs were restreaked on LB-agar plates with appropriate antibiotic, and then incubated in anaerobic conditions (a sealed plastic bag filled with nitrogen gas) for 16 hours in 37 degrees C. They were then taken out of the bag and placed under a camera that took pictures every five minutes over a 96 hour time span in room temperature. By analyzing several areas of each of the mutants in the pictures converted to grayscale, trendlines for their maturation were attained. This part, the purple amilCP, has a slower maturation time than the wild type blue amilCP.

References

Ahmed FH, Caputo AT, French NG, Peat TS, Whitfield J, Warden AC, Newman J, Scott C. 2022. Over the rainbow: structural characterization of the chromoproteins gfasPurple, amilCP, spisPink and eforRed. Acta Crystallographica Section D, Structural Biology 78: 599–612.

Liljeruhm J, Funk SK, Tietscher S, Edlund AD, Jamal S, Wistrand-Yuen P, Dyrhage K, Gynnå A, Ivermark K, Lövgren J, Törnblom V, Virtanen A, Lundin ER, Wistrand-Yuen E, Forster AC. 2018. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering 12: 8.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]