PROOF OF CONCEPT

The reasons for our endeavor to develop a biocontainment system for LMO co-culture with AHL concentration control, rather than single-culture containment, can be broadly categorized into two:

1. Maximizing the benefits of Modular Co-culture Engineering (see Description) while ensuring biosafety for versatile applications across various fields and industries.
2. Optimization for Single LMO biocontainment.

We believe the introduction of a quorum sensing system into the biocontainment system is the most suitable approach. AHL (C8-HSL), used in quorum sensing, functions as a population-regulating signal molecule in bacteria. It was chosen for the kill switch in biocontainment due to its minimal impact on yeast and microalgae growth. As AHL is specific to certain bacterial species, it operates strongly only in those, making it highly applicable for a specific biocontainment system.

To validate our concept in the real industry, we have developed a proof of concept in two specific parts:

• Validation via experts and market related investors

We found that one of the most active applications of co-culture is ironically in wastewater treatment plants. In these plants, thousands of microorganisms coexist in the bioreactors, breaking down various organic compounds. This includes the yeast we are researching. Through entrepreneurship competitions, support programs, and the guidance of mentors and investors with extensive experience in the environmental sector, we confirmed the significance of GMO regulation and biocontainment (see Humanpractice overview). With the information we gathered, our dry lab designed a film for microbe attachment and growth and an automated bioreactor (see dry-lab).

• Experimental and application validation

To create a quorum-sensing biocontainment system, we incorporated AHL as a concentration-dependent kill switch. We combined the quorum sensing system with a toxin-antitoxin system, breaking it into two parts: 1) Inducible-irreversible genetic switch (see Part 1.) and 2) Designing Cross-kingdom communication (see Part 2.) that varies the expression of antitoxin based on AHL molecule concentration.