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"The only thing to do with good advice is to pass it on. It is never of any use to oneself." - Oscar Wilde

Our team is very thankful for all the knowledge and advice bestowed upon us by scientists, farmers, and fellow iGEM teams. Pleasantly, we share what we have learned and created with the hope that they may serve as a resource for future iGEM teams to draw upon and benefit from.

New parts for biosafety

Biosafety serves as not merely a component, but the very foundation of sustainable synthetic biology. While numerous iGEM teams have diligently crafted various genetic elements to enhance the safety of their products, including auxotrophies, toxin/antitoxin systems, and barcode detection systems, our team wanted to go the extra mile. First, we developed a DNA-barcode detection method in which the new part BBa_K4663000 is stably integrated into the genome of the host organism, ensuring the traceability of our bacterium at every moment. Then, we integrated a kill-switch that aims to remove the entire genetic information of the bacterium upon induction.

As a novel function, we combined the two facets of biosafety – the DNA Barcode and the CRISPR/SuCas12a2 system. Our CRISPR/SuCas12a2 system will be activated upon expression of our DNA-barcode, which in turn is induced after addition of L-rhamnose to the soil. In this way, we aim to limit horizontal gene transfer by degrading the genetic material of our bacteria. We are convinced that by doing so, we add an extra level of biosafety to the bacterium that we are intending to release to the environment.

Successful iGEM teams, like the one of Copenhagen, 2022, want to apply their genetically engineered bacterium to the environment. For those and similar projects, it will be interesting to adopt our system and combine the traceability and removal of genetic information in a single biosafety system.

You can find more information about these parts on our biosafety wetlab in the results. To see a video about how farmer Herman applies our barcode detection method on his orchard, visit our applied design page!

Novel live biosensor for root delivery applications

The aim of our project was to develop a bacterium that could effectively deliver plant hormones to fruit trees upon attachment to their roots while minimising the energy burden imposed on them. To achieve this, we have re-designed biosensors from previous iGEM teams (WageningenUR, 2021) to respond to root exudates, which will be useful for future iGEM teams working with bacteria that target roots.

Our biosensor uses a toehold switch as described for the part BBa_K1586000, integrating signals from root exudates and quorum sensing. Although we constructed our biosensor using pre-existing components the synergy of these parts consider the specific environment of the soil to which our bacterium will be applied. We conducted extensive measurements to explore which inducible expression system is best suited for sensing root exudates.

Future iGEM teams that work on bacteria living in the rhizosphere can build upon our findings and use our toehold switch system to guarantee that their target protein is only expressed when required.

Explore our toehold switch in the secretion wetlab page!

New protocols for genetic engineering of Pseudomonas fluorescens

One of our primary goals was to conduct our experiments using a chassis that is well-suited for the environment where our project would eventually be applied. We identified Pseudomonas fluorescens strains SBW25 and etHAn as ideal candidates, fully aware that genetic engineering protocols for these strains are not widely available.

We developed and optimized transformation protocols for P. fluorescens. Within the "protocol" section, future iGEM teams will find comprehensive instructions on performing electroporation, heat shock, and conjugation transformations with the selected Pseudomonas fluorescens strains.

Future iGEM teams that aspire to work with P. fluorescens will benefit from this database, hopefully making their transformations successful from the very first trial!

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