Throughout the course of our project, there were many previous iGEM teams that have guided and contributed to our inspiration. A notable team that has aided in our project is UCalgary’s 2017 iGEM team. Their work on the PHB synthesis pathway allowed us to focus on the TPA degradation pathway and connect the two pathways for plastic bioconversion.
The TphA123, TphB and TphC enzyme sequences were taken from the genome of Comamonas sp. Strain E6 by (Sasoh et al. 2006). The sequences that were optimized for expression in E. coli using the online “Codon Optimization Tool” by IDT. These optimized sequences were integrated into our TPA degrading plasmid construct. Our team is using enzymes that were discovered in the bacterial strain of Comamonas sp. for a novel application by incorporating them into a pathway used for the degradation of PET plastics. This could be built upon in the future by optimizing the expression or possibly engineering the enzymes to be more catalytically active.
Originating from Comamonas sp. E6, the integral components, TpiA and TpiB, make up the large and small transmembrane proteins, respectively. These proteins play a pivotal role within the context of our detection system, as interact with TphC, to encode for a tripartite transporter essential for the uptake of TPA. The basic parts provide a foundation to be built upon, where the expression and activity of the transporters can be measured and optimized.
Through the incorporation of the TphR protein within our repurposing system design, we can potentially induce the transcription of the TPH degrading genes when only in the presence of terephthalate, and we used this characteristic to design a biosensor. The use of the TphR transcriptional regulator in the future could allow for other teams to improve upon this part or possibly find a new application for its use, such as for the regulation of expression of TPA degrading enzymes, to make the process more energy efficient.
The composite part BBa_K4728016 provides a building block for future iGEM teams to engineer the TPA-degradation pathway into their chosen chassis. In addition, BBa_K4728021 (TPA detection device using tripartite transporter) and BBa_K4728022 (TPA detection device with the transporter TpaK) can provide following teams with a TPA biosensor that can provide visual indication of TPA concentration. This can be used to detect PET degradation (increase in TPA) or TPA degradation (decrease in TPA concentration). The composite parts also serve as a model for future teams to potentially base their TPA-related composite parts off, whether it be for a biosensor or to regulate protein expression with TPA inducers. It may also serve as a model to design other aromatic acid induced composite parts.
We submitted a basic part for the PhaF CDS (BBa_K4728007) as well as an improvement of the part (BBa_K4728008), which has higher binding affinity for PHB. This was partially inspired by iGEM 2017 Calgary’s work on PHB secretion with phasin, who suggest improving phasin’s binding affinity can improve the production and secretion of PHB. Now that our improved part has been tested in silico, future teams can use the part for their project and test in vivo.