Contribution
Overview
Hoping to make a useful contribution for future iGEM teams,we completed the experimental characterization of existing parts tsPurple, purple chromoprotein(incl RBS)(BBa_K1033905) and meffBlue, blue chromoprotein(BBa_K1033901)and provided new documents for both of them.
The details are in the part registrations pages linked in the table below.
New documentation to existing Parts
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.
References
1. 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.
2. 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.