Part:BBa_K2510000:Experience

All four coral acid-rich proteins(CARP1-CARP4) were implemented into the pSB1C3 expression vector. Sequencing was performed to confirm the construction. The expression vectors were then transformed into Escherichia coli BL21. The colonies with an appropriate plasmid construct were grown in LB/chloramphenicol medium, LB/CaCl, LB/ASW and CARPs were expressed by induction with 0.2% L-arabinose and 1mM IPTG.

Figure 1:SDS-PAGE separated by 10% SDS-PAGE CARP1-CARP4 proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field and stained by Coomassie Blue.

The size of expressed proteins was checked by running at 10% SDS-PAGE gel. After extracted with BugBuster extraction reagent proteins were placed in artificial seawater and CaCl solutions to check on precipitation. Precipitated material was stained with 1.5% Alizarin Red S dye mixed with 0.5% HCl. The reaction between carbonates and acid is usually controlled so that the more reactive minerals, such as calcium carbonate and aragonite, stain dark red close to the black but the less reactive ones, such as dolomite and siderite, remain unstained.

Figure 2:Alizarin Red staining for detection of calcium carbonate composites in the precipitated proteins: A) stained calcium carbonate powder - positive control, B) stained sample of BL21 extracted protein precipitation in CaCl 1M solution - negative control , C) stained sample of CARPs extracted protein precipitation in CaCl 1M solution. "

Figure 3: Stained calcium carbonate deposits formed in the present of CARPs in artificial seawater(ASW)

Applications of BBa_K2510009

The positive aspect of using CARPs proteins is that we can increase the production of microbial calcium carbonate by reducing its sensitivity to changes in the level of acidity in the environment, as well as get rid of side products in our system such as urea. Furthermore, this is a one enzyme pathway, allowing the cost of the cells to be greatly reduced.

User Reviews

1. Tali Mass, Jeana L. Drake, Liti Haramaty, J. Dongun Kim, Ehud Zelzion, Debashish Bhattacharya, and Paul G. FalkowskiCurrent, Cloning and Characterization of Four Novel Coral Acid-Rich Proteins that Precipitate Carbonates In Vitro, Biology 23, 1126–1131, June 17, 2013.

2. Stanislas Von Euw et all., Biological control of aragonite formation in stony corals, Science 02 Jun 2017.

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