Part:BBa_K2387001

Monomeric chromoprotein anm2CP controlled by inducible araC/pBAD promoter

In this composite, the coding sequence for anm2CP is under the control of the inducible pBad/araC promoter (BBa_I0500), as well as a strong RBS (BBa_B0034).

Usage and Biology

anm2CP is a monomeric chromoprotein (BBa_K2387000), the only one that is known to be produced in nature. The sequence belongs to the jellyfish Anthomedusae sp. SL-2003 (GenBank, AY485336). It shows a bright pink color when expressed to sufficient concentrations (Figure 1). Its absorbance spectrum shows a pick with a maximum at 572 nm. As other fluorescent proteins and chromoproteins, it has a structure similar to that of GFP, as it can be observed from the structure of KillerRed ([http://www.rcsb.org/pdb/explore.do?structureId=2wiq PDB 2WIQ]) a fluorescent protein evolved from anm2CP. Most chromoproteins are tetrameric, which makes them a bad candidate for protein fusions and other applications. Among other applications, anm2CP would be able to be split in order to be used in bimolecular complementation techniques.

Figure 1: On the left, E. coli BL21 without plasmid. On the right, E. coli BL21 expressing anm2CP induced by 0.2% arabinose.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1280
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Absorbance Parameters

The spectrum of absorbance for anm2CP was determined (Figure 2). It shows a clear pick with a maximum of absorbance at 572, which grants the chromoprotein the bright pink color. The spectrum is compared to that one of BL21 without plasmid (Figure 3), which serves as a negative control. The spectrum of E. coli BL21 was subtracted from the spectrum of anm2CP to remove the effect of other proteins and impurities(Figure 4). The comparison in the absorbance at 572 nm shows a clear difference between E. coli BL21 without plasmid and E. coli BL21 expressing anm2CP (Figure 5). This difference can be used to compared different levels of functional anm2CP in the cell.

Figure 2: Absorbance spectrum of anm2CP

Figure 3: Absorbance spectrum of empty E. coli BL21.

Figure 4: Absorbance spectrum of anm2CP corrected with the spectrum of empty E. coli BL21.

Figure 5: Absorbance difference at 572 nm between empty E. coli BL21 and E. coli BL21 expressing anm2CP.

Methods

• Incubate cells overnight with chloramphenicol and 0.2% Arabinose.
• Lyse cells using a method that does not denature proteins (e.g. enzymatic lysis or sonication).
• Measure absorbance in lysate

References

• Dmitry A. Shagin, Ekaterina V. Barsova, Yurii G. Yanushevich, Arkady F. Fradkov, Konstantin A. Lukyanov, Yulii A. Labas, Tatiana N. Semenova, Juan A. Ugalde, Ann Meyers, Jose M. Nunez, Edith A. Widder, Sergey A. Lukyanov, Mikhail V. Matz. (2004) GFP-like Proteins as Ubiquitous Metazoan Superfamily: Evolution of Functional Features and Structural Complexity, Molecular Biology and Evolution, 21(5) 841–850. https://doi.org/10.1093/molbev/msh079
• Bulina, M. E., Chudakov, D. M., Britanova, O. V, Yanushevich, Y. G., Staroverov, D. B., Chepurnykh, T. V, … Lukyanov, K. A. (2006). A genetically encoded photosensitizer. Nat Biotech, 24(1), 95–99. http://dx.doi.org/10.1038/nbt1175