Part:BBa_K1033900

meffBlue, blue chromoprotein (incl RBS, J23110)

This chromoprotein from the coral Montipora efflorescens, meffBlue (also known as Rtms5), naturally exhibits strong color when expressed. The protein has an absorption maximum at 592 nm giving it a blue 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 24-64 hours of incubation. The protein meffBlue has significant sequence homologies with proteins in the GFP family.

Usage and Biology

This part is useful as a reporter.

Contribution

Group: Linkoping_Sweden iGEM 2021
Author: Ewelina Bladh
Summary: In this contribution, we recorded color development in colonies of E.coli cells of strain XL1. In addition to studying the color development, we determined meffBlue's absorbance maximum wavelength.

Color Development
In order to determine the progression of color development in colonies, E.coli cells (XL1) were plated onto LB-Miller agar plates containing chloramphenicol. The cells were plated in two ways. The first by plating 100 uL immediately from the liquid culture. The second by first centrifuging the culture, discarding 90% of the supernatant and resuspending the cell pellet in the remaining 10%, followed by plating 100 uL of the resuspension. This results in what is from now on referred to as the diluted and concentrated plate, respectively.

These plates were incubated in 37C for the following four days, where they were checked upon each day. Figure 1, displayed below, shows the diluted and concentrated plates during this four day study. The illustration of the diluted plate shows that after 12 hours, no color can be seen to the naked eye. This changes over the course of the next 12 hours, where at 24 hours the colonies are clearly visible. (doublecheck hours) The color intensity increases over the next two days. The same can be said about the concentrated plate, but unlike the diluted plate the concentrated plate shows indications of colored colonies already at 12 hours.

Figure 1. Top shows the diluted plate over the course of four days. Bottom image shows the concentrated plate over the course of four days. The time each picture was taken after plating is displayed under the each picture in hours.

Absorbance

Source

Montipora efflorescens. The protein was first extracted and characterized by Beddoe et. al. under the name Rtms5 (UniProtKB/Swiss-Prot: P83690.2). This version is codon optimized for E coli by Genscript.

References

[http://scripts.iucr.org/cgi-bin/paper?cy0089] Beddoe, Travis, et al. "The production, purification and crystallization of a pocilloporin pigment from a reef-forming coral." Acta Crystallographica Section D: Biological Crystallography 59.3 (2003): 597-599.

[http://www.sciencedirect.com/science/article/pii/S0969212603000285] Prescott, Mark, et al. "The 2.2 Å crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation." Structure 11.3 (2003): 275-284.

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]