Microbial Biocontainment via Nutrient Dependency and Recombinase Control
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current biocontainment technologies for microorganisms rely on single mechanisms, such as auxotrophic mutations or toxin-antitoxin pairs, which compromise fitness and are prone to leakiness, making them unsuitable for industrial applications and increasing the risk of escape mutants, while also being costly for large-scale use.
Innovation Solution
Development of modified microorganisms that require exogenously provided compounds for growth, using a combination of inducible promoters and site-specific recombinase systems to control genetic alterations, ensuring viability without adverse effects on fitness, and incorporating decoy compounds to maintain gene expression profiles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If auxotrophic mutations or toxin-antitoxin pairs are used for biocontainment, then microbial growth can be controlled, but fitness is compromised and normal behavior is affected
Solution Approach 1:
The patent changes the concentration parameter of a nutrient (e.g., amino acid, vitamin, or other growth factor) from normal levels to sub-micromolar levels. This parameter change creates a dependency on exogenous compound provision without requiring genetic modifications that compromise fitness. The microorganism maintains normal growth and behavior when the compound is supplied at the optimized sub-micromolar concentration, while growth is inhibited when the compound is removed.
Solution Approach 2:
The patent replaces the mechanical/genetic biocontainment systems (auxotrophic mutations, toxin-antitoxin pairs) with a chemical control mechanism based on nutrient dependency. Instead of using genetic circuits or protein-based containment systems that inherently reduce fitness, the invention uses a simplified nutritional dependency achieved through sub-micromolar compound provision, which does not adversely affect microbial fitness or normal behavior.
2Device complexity
If single mechanism biocontainment is used, then implementation is simple, but leakiness occurs and escape mutants arise
Solution Approach 1:
The patent segments the biocontainment strategy into multiple independent layers: (1) sub-micromolar nutrient dependency, (2) optional inducible promoter control, and (3) optional site-specific recombination control. This segmentation provides redundancy and reduces leakiness without requiring complex integrated systems. Each layer independently contributes to containment security, and the modularity allows for flexible implementation based on specific application needs.
3Reliability
If auxotrophic strains are used, then biocontainment is achieved, but supplementation costs increase for industrial scale-up
Solution Approach 1:
The patent changes the concentration parameter from micromolar (traditional auxotrophic supplementation) to sub-micromolar levels. This parameter change dramatically reduces the quantity of compound required for supplementation, making the system economically viable for industrial scale-up while maintaining effective biocontainment. The sub-micromolar concentration provides sufficient control for containment security at a fraction of the cost of traditional auxotrophic supplementation.
Data Source
AI summary
Provided are modified microorganisms which are modified such that their growth can be controlled using exogenously provided compounds. The microorganisms can be modified by genetic alterations that include a promoter inducible by a first exogenously supplied compound. The promoter can be configured to drive expression of an RNA coding sequence that may be essential to growth of the microorganism. The microorganisms may also be modified to include site specific recombinase recognition sites flanking or within the RNA coding sequence so that expression of the corresponding site specific recombinase will disrupt transcription of the RNA. The site specific recombinase can be configured such that it expression and/or activity is suppressed by a second exogenously supplied compound. Methods of making the modified microorganisms and kits that contain reagents for making and using the modified microorganisms are also provided.