Difference between revisions of "Part:BBa K1033902"

Revision as of 16:11, 11 October 2022

meffBlue, blue chromoprotein

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.

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.

Result

meffBlue, blue chromoprotein This chromoprotein from the coral Montipora efflorescens, meffBlue (also known as Rtms5), naturally exhibits strong color when expressed. tsPurple, purple chromoprotein(incl RBS) This chromoprotein (also known as TinselPurple) naturally exhibits strong color when expressed. The chromoproteins came from the high copy plasmid pSB1C3 from the promoters J23116 and J23110. Our team was very interested in the characterization of these two proteins and carried out the design and implementation of related experiments.

In order to test the function of these chromoproteins, we constructed“Pet-29a（+）-tsPurple”and “Pet-29a(+)-meffBlue”. “Pet-29a(+)”is a commonly used prokaryotic expression vector of fusion protein type, which contains kanamycin resistance gene. The expression was induced by T7 RNA polymerase provided by host cells. E.coil BL21 was used as receptor bacteria to express the protein, and then the fusion protein with histidine was purified by Nickle-affinity chromatography. If these chromoproteins are functional, we can observe the correct band through plasmid PCR, agarose gel electrophoresis, and the obvious color of the colony. After a series of purification operations, the correct band was observed by SDS-PAGE. Finally, use Mass Spectrometer to detect the exact molecular weight of the protein for final confirmation. Our experiment results matched the general expected trend and data.

Sequence and Features