Team:UrbanTundra Edmonton/Design
Design
Our part is comprised of three gBlock sequences that work in tandem to reduce perchlorate ions at a controllable rate: PcrA, PcrA/B, and LacIq. Diagrams for the gBlocks are shown below in both a graphical representation as well as their respective genetic sequences.
Figure 1: Schematic for our Pcr construct
PcrA and PcrA/B
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. As well, the entire PcrA sequence, which was around 4000 bp, was too large to synthesize into one gBlock. The end part of the PcrA sequence is found on the PcrA/B gblock. The standard iGEM suffix was included at the end of the PcrA/B gBlock for compatibility with the PSB1C3 submission vector.
Figure 2: PcrA gBlock sequence
Figure 3: PcrA/B gBlock sequence; end part of PcrA is included in this sequence
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/B 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 LacIq gBlock will run in the reverse = 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 LacIq 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-IIS, 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 for use with our ligation method.
Figure 4: LacIq gBlock sequence; promoter reads in the reverse direction
References
Bender, Kelly et. al. “Identification, Characterization, and Classification of Genes Encoding Perchlorate” Journal of Bacteriology. vol. 187, no. 15, Mar. 2005. pp. 509-5093 . American Society for Microbiology, 10.1128/JB.187.15.5090–5096.2005
