Design

Our part is comprised of three gBlock sequences that work in tandem to reduce perchlorate ions at a controllable rate. Diagrams for the gBlocks are shown below in both a graphical representation as well as their respective genetic sequences.

PcrA and PcrB

In the original work of Bender et al., the perchlorate reductase (Pcr) gene in two Dechloramonas bacterial species were identified, characterized, and classified. It was found that the Pcr gene was a membrane-bound protein capable of converting ClO4- into ClO2- and O2 and that it was comprised of multiple subunits, where the subunits PcrA and PcrB made up the functional subunits of this system. We took the wildtype gene sequences for these two subunits and designed them for functionality within an E. coli chassis. Both sequences are included within a single operon to ensure that they are both expressed in the proper stoichiometric proportions. At the beginning of this operon is a promoter region, which itself is flanked by Operator 1 sequence downstream and Operator 2 sequence upstream. These operators were taken from the Tinsel plasmid and work with the LacI gBlock, which will be discussed below. Ribosomal binding sites were added at the beginning of each subunit sequence to promote ribosome recruitment during translation. The natural sequences of PcrA and PcrB contained a number of unwanted restriction sites, so we introduced silent mutations at these sites to remove them. The standard iGEM suffix was included at the end of the PcrB gBlock for compatibility with the PSB1C3 submission vector.

LacI

The addition of the LacI gBlock introduced a way to regulate the production of the Pcr enzyme. This sequence codes for the LacI repressor protein. When this protein is expressed, it will latch onto the operator sites found beside the promoter region of the PcrA/PcrB operon; this prevents the transcription of PcrA and PcrB, inhibiting the expression of the Pcr gene. This repressor can be removed by inducing them with IPTG, allowing promoters to read and copy the Pcr operon. The promoter for the LacI gBlock will run 3’ to 5’, opposite to the direction of transcription of PcrA and PcrB, reducing the chance of overexpression of the Pcr enzyme. Lastly, the standard iGEM prefix was appended to the beginning of the LacI gBlock so that it can be inserted into the PSBC13 vector.

Additionally, BsaI restriction sites were included at points along the gene sequences of PcrA and PcrB. Since the BsaI restriction enzyme is Type 2-S, the enzyme will cleave the DNA strand off-site. By customizing the areas that are cut by BsaI, we can facilitate the ligation of the gBlocks in the proper order using our ligation method.