Part:BBa_K2179003
Gene Design
Chlorite Dismutase is the second enzyme utilized in a number of bacterial species' perchlorate reducing pathway. In their original work, Thorell et al. cloned, sequenced, and functionally expressed the natural coding sequence for Chlorite Dismutase from I. dechloratans. They determined that the gene was capable of converting ClO2- ions to O2 gas and Cl- ions and that its activity was localized to the periplasm, even though its putative signal peptide had not been cleaved. We designed this Cld(-SP) gene to lack this putative signal sequence. The purpose of this modification was so that once translation was complete, the Chlorite Dismutase enzyme would remain in the cytoplasm of our E. coli. chassis. A C-terminal histidine tag was also to this translational unit in order to purify the enzymes in the event that O2 production from living cells proved inefficient. Flanking these BsaI sites were XbaI (5’) and the BBa_ std 1 suffix (3’) to facilitate parts creation for the iGEM Registry. We note that the natural coding sequence contained none of the standard BBa_10 restrictions sites, but did contain a single internal BsaI site which we eliminated with a single base silent substitution.
Functionality
An oxygen production assay was conducted where transformed E. coli was combined with solid Sodium Chlorite. Almost immediately, the formation of a gas, which was inferred to be oxygen, was noted. This indicates the proper functioning of our synthetic G-Block and aligns with the results of Thorell et. al.
References
Thorell, H.D, Karlsson, J., Portelius, E.and Nilsson, T. Biochimica et Biophysica Acta 1577 (2002) 445–451 Samant,S., Gupta, G., Karthikeyan, S., Haq, S., Sambasivam, N.G., and Sukumaran, S. J Ind Microbiol Biotechnol (2014) 41:1435–1442
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