A new method called LySE overcomes key limitations in gene engineering, allowing scientists to rapidly evolve large gene clusters with unprecedented control.
Researchers at the National University of Singapore (NUS) have developed LySE (Lysis-based Selection and Evolution), a continuous evolution method that addresses the limitations of existing phage-assisted continuous evolution (PACE) technologies.
LySE can handle gene clusters up to 40 kilobases in length — five times the limit of PACE — and crucially, it avoids the problem of "cheater" mutations in the host bacteria.
How LySE Works
- The System: LySE uses a modified bacteriophage T7 that infects E. coli bacteria. The phage packages a phagemid (a small DNA ring) carrying the target gene cluster into new virus particles.
- Mutagenesis: An engineered, error-prone T7 DNA polymerase introduces mutations into the phagemid during replication. This polymerase has a mutation rate about 160,000 times higher than the bacterium's own DNA copying system.
- Controlled Evolution: The high error rate also introduces mutations in the phage’s own DNA, weakening the phage and limiting its ability to spread uncontrollably. By adjusting the phage-to-bacteria ratio, researchers can toggle between a mutation phase (where target genes accumulate mutations) and a selection phase (where mutated genes are tested for improved function).
Performance Validation
- Antibiotic Resistance Test: In a test for antibiotic resistance, the improved trait persisted after transferring the genes to new bacteria, confirming that changes were confined to the target gene cluster.
- Ethylene Glycol Pathway: LySE was tested on a five-gene pathway that enables bacteria to use ethylene glycol for growth. After five rounds of evolution with decreasing glucose, the best-performing strain produced 50.9% more biomass using ethylene glycol as the sole food source.
- Mutation Confirmation: Sequencing revealed that LySE introduced mutations in both regulatory regions and protein-coding genes. Each beneficial mutation was confirmed by adding it back individually into a fresh host.
Advantages and Applications
"LySE confines improvements to the target gene cluster, avoiding unwanted mutations elsewhere in the bacterial genome, and allows easy transfer of optimized pathways into different bacteria."
The method requires only standard laboratory equipment and the mixing of phage lysates with cell cultures, making it accessible to laboratories without specialist phage biology expertise.
Potential applications include:
- Optimizing biosynthetic pathways for pharmaceuticals
- Engineering microbes for environmental pollutant degradation
- Evolving synthetic metabolic routes for carbon capture
A patent has been filed for LySE. The team plans to apply it to entirely synthetic systems, such as optimizing computationally designed CO₂-fixing metabolic pathways for efficient function inside living cells.