Introduction

At Pyre we hope to advance the development of whole cell bioremediation research by providing a stepping stone for future iGEM teams. Our characterization of new basic parts involves an anchor protein which can be used for surface expression of any enzyme, which is fitted to a linker and repeater sequence that improves flexibility and integration of any membrane enzyme. Our successful development of aptamer-based biosensor and its quick adaptation to a second target, highlights the versatility of this approach, laying the groundwork for a new class of biosensor development with well documented protocols and troubleshooting. Our novel computational modelling tools allow future teams to better consider growth yield for their proteins ensuring they choose the optimal promoters and copy numbers. Our spatial model also allows teams to consider how their chassis would spread over a region, allowing them to better consider biosafety and deployment.

New Anchor Protein Basic Part

While Pyre1 was developed specifically for the degrading λ-cyhalothrin – and has been added to the database for replication and refinement by other teams – it was always the plan to create a modular system that could be easily adapted to as many different pesticides as possible. After discovering that the addition of a specific repeater sequence to a surface-expressed protein could increase the likelihood of membrane insertion, we decided that a basic part consisting of the conserved sequences of the repeater sequence, linker and anchor protein INP-N was worth having. The basic part was submitted under accession number BBa_K4210005.

Novel Aptamer Biosensors

After running several cycles of optimisation tests via intense troubleshooting, we were able to develop an aptamer-based biosensor which is able to indicate the presence of our target pyrethroid pesticide, λ-cyhalothrin. The versatility of our biosensor was evidenced by a successful adaptation of our λ-cyhalothrin biosensor to target an organophosphate pesticide fenitrothion instead, which was a part of our partnership agreement with the Concordia University iGEM 2022 team, CyanoClean.

The protocol for the development of these aptamer-based biosensors has the potential to be easily adopted to future iGEM teams considering a biosensor component within their project, as the system only requires a simple change involving the aptamer sequences and the target compound for the sensing system to work. The full protocol can be viewed here. Subsequently, our optimised aptamer-based biosensor would serve as a starting point for future iGEM teams in generating their personalised sensing system. Specific aptamer sequences could be obtained through SELEX or prior work.

Computational Modelling and Overall Support

To explore growth-production trade-offs, we developed an ordinary differential equation model of microbial gene expression and growth. This model can be freely used by future iGEM teams to optimise their degradation rates, as consecutive promotion is not always the best choice. Our spatial model also allows teams to consider how their chassis would spread over a region, enabling them to better consider biosafety and deployment. Our holistic human practices model which maps out stakeholders and the opposing forces within will accelerate future iGEM teams in identifying their connections to the real world and where they place themselves in the highly intertwined economy. We aim to make all our resources and learnings throughout iGEM available in a handbook for future teams, to best support future iGEM teams in their endeavours.