We’ve done the groundwork. Feel free to build on it!
Like our project, our contributions can be split into three parts:
With the expression part, we contribute several laccase genes from different fungal species which are codon optimized for expression in Pichia pastoris:
We optimized P. pastoris growth conditions and media compositions for potential laccase production.
Furthermore, we advanced the laccase analysis techniques. We showed that with our ABTS-Assay we can detect laccase activity even before the protein can be detected using SDS-PAGE with silver staining.
In the immobilization part we tested the method of enzyme agar immobilization - a simple and cheap method which has given us promising results. Not only can this method be used to immobilize enzymes, but our results have also shown that it can be used to enable enzymes to be active at a broader pH range . We encourage other iGEM teams to further explore the possibilities of this method.
Finally, through our research we found possible yeast promoters which could respond to Diclofenac and/or Ibuprofen levels in the yeast’s surroundings. Although we were not successful in developing our biosensor, our work may serve as a foundation for future iGEM teams aiming to achieve this objective.
The promoters we submitted to the iGEM registry:
While we encountered some challenges in achieving high levels of laccase expression and activity, our work provides valuable insights into the process of heterologous laccase expression in P. pastoris. This groundwork could be useful for future iGEM teams working on similar enzyme expression projects in yeast systems.