Contribution

1. Temperate Phage Therapeutics: Genetic Engineering that Beyond Antibiotics

In the face of escalating antibiotic resistance, our project embarked on an expedition to unravel alternatives solutions for curbing bacterial infections. Through extensive literature review and consultations with field experts, we fully realized the burgeoning significance of phage therapy. Temperate phages furnish a unique scaffold for genetic engineering endeavors aimed at precise biofilm disruption. Their competency in integrating into the host genome opens the gateway to introduce genetic constructs capable of thwarting biofilm formation and antibiotic resistance machineries. Through this exploratory lens, our project not only elucidates the comparative merits of temperate phage therapy, but also propels the frontier of genetic engineering in combating antibiotic resistance, thereby contributing a novel perspective to the microbial warfare narrative.

2. Deciphering Biofilm Dynamics: The Game-Changing Potential of aiiA Gene Integration

Following an exhaustive review of the literature, we initially targeted four genes, wspF , yhjH , aiiA , and ytnP , for editing. Employing the introduction of high-copy-number plasmids for indirect validation, coupled with the elimination of the pf4 sequence for verification, we demonstrated the feasibility of all four genes as biofilm repressing elements. However, due to the high failure rate of homologous recombination, successful integration into the genome was achieved only for aiiA and yhjH . Through a series of molecular assays and biofilm formation experiments, we substantiated that the insertion of aiiA significantly curtails biofilm formation.

This investigative venture not only illuminates the potential of strategic gene editing but also accentuates the pivotal role of aiiA in biofilm dynamics. Our findings contribute to a broader understanding of microbial interactions and present a promising avenue for combating persistent biofilm-associated infections. Through meticulous genetic manipulation and experimental validation, our work epitomizes the confluence of synthetic biology and microbial ecology in devising innovative solutions for real-world microbial challenges.

3. Modeling Microbial Dynamics: Elucidating the Power of Gene Interference in Pseudomonas aeruginosa

To sum up, we developed three models. Firstly, we constructed a population growth model for P. aeruginosa using growth differential equations to calculate the concentrations of bacteria and viruses over time. This model elucidated the required burst size and infection rate for the virus to rapidly infect the entire colony. Subsequently, we conducted flux balancing analysis on a modified P. aeruginosa metabolic model by incorporating reactions related to the cyclic-di-GMP pathway. Simulation results showed that insertion of yhjH and wspF genes significantly inhibited c-di-GMP levels and biofilm formation. Finally, we constructed another differential equation model to investigate the impact of insertion of aiiiA and ytnP genes on quorum sensing in P. aeruginosa . By calculating critical points of the equations, our experiment demonstrated that the insertion of these genes significantly increased the activation threshold of quorum sensing and decreased its releasing threshold. Overall, our models support the notion that the insertion of our selected genes significantly impairs biofilm formation in P. aeruginosa , suggesting that mild bacteriophage therapy may serve as an effective treatment method.