Adaptive Laboratory Evolution Setup

The challenge of toxicity and the solution with Adaptive Laboratory Evolution

Adaptive laboratory evolution (ALE) is a powerful method in metabolic engineering and molecular biology, leveraged to enhance microbial strains' performance by exploiting beneficial mutations under environmental stress conditions. As an example, precision fermentation relies heavily on microbial strains such as Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae or cerevisiae), aiming to produce high-value products for pharmaceuticals, biofuels, and new plastics, but the developed strains often face toxicity limitations that cap the yield of their production. Adaptive laboratory evolution is one of the key techniques used to reduce this sensitivity and increase the overall yield achievable with modified strains.

MilliDrop's AzurEvo platform transforms adaptive laboratory evolution by providing an advanced solution for efficiently evolving microbial strains under selective environmental stresses. The combination of reduced consumption, accelerated mutation detection, and enhanced experimental reliability positions AzurEvo as a valuable tool in modern synthetic biology and metabolic engineering, facilitating quicker development and commercialization of bio-based products.

Limitations of Traditional Adaptive Laboratory Evolution

Typical ALE experiments, frequently discussed in journals like ACS Synthetic Biology, Springer Nature, and Environmental Science, involve chemostat cultures or flask-based cultures. Traditional methods present critical challenges:

• High Substrates Consumption: Substantial usage of rare compounds increases costs and limits experimental scale.

• Risk of Population Collapse: Abrupt increases in environmental stress can eliminate microbial populations, requiring repeated experimental evolution cycles.

• Lengthy Evolutionary Engineering Cycles: Extended experimental timelines hinder rapid development of tolerance to stressful metabolites.

The AzurEvo Platform: Enhancing Laboratory Evolution with droplets

MilliDrop's AzurEvo platform significantly advances laboratory evolution protocols, optimizing adaptive laboratory evolution through droplet microfluidics. Each droplet functions as an independent micro-reactor, enabling simultaneous experimentation across numerous concentrations of stressors.

How the AzurEvo Platform Streamlines Adaptive Evolution

• Precise Adaptive Evolution: Extensive parallel testing across around 95 concentrations with multiple replicates provides substantial source data and supplementary information.

• Rapid Mutation Detection: Real-time growth tracking enables quick identification of adaptive mutations and key mutations contributing to stress resistance.

• Minimized Supplementary Data Requirements: Droplet technology efficiently generates robust experimental evolution data, significantly reducing supplementary data management complexities.

Step-by-Step Adaptive Laboratory Evolution with AzurEvo

1. Droplet Generation: From a precise formulation of microbial cells, media, and targeted stressor (e.g. resistance-inducing compounds).

2. Monitoring: Continuous tracking to rapidly detect beneficial mutations and growth signaling evolutionary adaptations.

3. Evolved Strain Recovery: Automatic recovery of resilient microbial cells for further rounds of experimental evolution.

4. Iterative Mutation Enhancement: Iterative cycles rapidly intensify adaptive laboratory evolution, increasing microbial strain tolerance and genetic diversity.

Advantages Over Traditional ALE Experiments

• Reduced Molecule Consumption: Drastically lower amounts required compared to traditional methods.

• Faster Experimental Cycles: Rapid identification of growth shortens overall ALE experiment durations significantly.

• Increased Experiment Reliability: Independent micro-reactors protect against complete microbial population collapse, allowing for more daring approaches.

• High Statistical Power: Vast supplementary data and comprehensive source data from droplet arrays support robust statistical conclusions.

Applications: Expanding Laboratory Evolution into Practical Synthetic Biology

AzurEvo enhances ALE experiments for multiple fields:

• Antibiotic Resistance Studies: Rapid assessment and evolution of bacterial genomes for clinical trials.

• Biofuel Production: Accelerated development of stress-resistant microbial strains for increased production efficiency.

• Pharmaceutical Products Yield Development: Evolution of strains capable of producing therapeutics at commercially viable levels.

For example, ALE experiments involving E. coli or cerevisiae to produce bio-based aromatic compounds illustrate AzurEvo's ability to significantly reduce resource usage and enhance the speed of obtaining viable mutant strains.

Litterature:

• Dragosits & Mattanovich (2013), "Adaptive laboratory evolution – principles and applications," Microbial Cell Fact.

• Portnoy & al (2011), “Adaptive laboratory evolution — harnessing the power of biology for metabolic engineering”, Current Opinion in Biotechnology.

• Sandberg et al. (2019), "The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology," Sciences Direct.

Further ALE research can be explored extensively through Google Scholar or other platforms for comprehensive foundational knowledge.