Gene Design

Figure_. Shown is a schematic, of the natural chlorite (Cld) dismutase coding sequence from I. Dechloratans as originally characterized by Thorell et al. and the G-Block designs for Cld with (+SP) and without (-SP) the periplasmic localization signal peptide. In Cld(+SP) the signal peptide from the E. coli MalE gene replaces the the putative signal peptide of the natural gene. Both G-Blocks duplicate the RBS driving the Tinsel cassette of PCB-38-441 (Fig_) up to BsaI site. Both coding sequences have been appended with a stretch of 10 histidine residues at their C-terminus for Nickel column purification of each enzyme. The single BsaI site contained in the natural gene was eliminated by a silent mutation (see Fig.__). The Restriction sites flanking the Bsa I sites were included to create Parts according to BBa std 10.

CLD G-Block

CLD± G-Block designs are shown below in Figures_ and _ as schematics or sequences. In their original work, Thorell et al. cloned, sequenced, and functionally expressed the natural coding sequence for Cld from I. dechloratans.

Modifications

CLD±

Thorell determined that the gene was capable of converting ClO2 to O2 and Cl- and that its activity was localized to the periplasm, even though its putative signal peptide had not been cleaved. In light of their results we decided to design a Cld gene (Cld SP+) where the putative signal peptide was replaced with the MalE signal peptide from E. coli that has been previously shown to be a superior signal for periplasmic localization that is cleaved (Samant et al. 2014). In addition we designed a Cld gene that lacked the signal sequence (Cld SP-) as a negative control.

Terminal Histidine Tag

In the event that the production of O2 from living cells proved inefficient, a C-terminal histidine tag was appended to both the CLD- and CLD+ G-Blocks in anticipation for the purification the expressed enzymes. Nickel Column info and reference pls.

Restriction Sites and Silent Mutation

Flanking these BsaI sites were XbaI (5’) and the BBa_ std 1 suffix (3’) to facilitate parts creation for the 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 (Fig_)

IDT Synthesis

Upon the finalization of our CLD G-Block designs, we were able to get the gene synthesized by IDT who was offering iGEM teams 15 000 base pairs worth of DNA for free.