Programming Microbial Communities via Control of Plasmid Dynamics

You, Lingchong

Son, Hye-In

2024-06-06T13:44:06Z

2024

Biomedical Engineering

Cells can sense and respond to various environmental cues. In the past 25 years, this ability has been exploited in engineering many innovative applications, ranging from bioproduction and metabolic engineering, to living therapeutics and biosensing. Despite tremendous advancements in complex genetic circuit development, the field still suffers from several limitations. For instance, evolutionary pressure can hamper the long-term genetic stability and functionality of circuits. The long incubation times required for cell growth serve as a fundamental rate limiting step for routine microbiology experiments and circuit engineering. Available biological parts, such as promoters and ribosome binding sites, often confer a limited dynamic range of gene expression levels and are incompatible, exacerbating the construction of higher order circuits.

Plasmids are extrachromosomal DNAs, usually circular, that replicate independently of the host genome. Because they are easy to manipulate and engineer, plasmids have served as a popular workhorse for programming desired functions in microbial populations. Plasmids can maintain steady average copy numbers in hosts, and a specific plasmid type can be chosen to express genes at a desired level. However, recent studies have focused on the dynamic modulation of plasmid copy number as a new engineering strategy, which is still underexplored. Understanding plasmid dynamics can provide insights to harness powerful tools for engineering microbial communities and offer a new avenue to overcome the current challenges in synthetic biology.

In this dissertation, I used mathematical modeling and synthetic biology approaches to develop methods for engineering microbial communities by exploiting and manipulating plasmid dynamics. First, I examined the sources of circuit failure and studied design strategies for enhancing synthetic gene circuits’ stability in microbial hosts for robust long-term performance. I summarized the engineering strategies into two categories: (1) to suppress the chance of mutant emergence by reducing the evolutionary pressure; and (2) to suppress the relative fitness of mutants by selecting against genetic variants.

Applying some of the identified engineering strategies, I developed synthetic gene circuits, named Red Queen circuits, that can modulate the host cell viability according to its growth rate. Using the circuit, I achieved a 250% increase in host cell growth rates at the end of a 100-day long-term adversarial laboratory evolutionary experiment, during which the circuit continuously suppressed slow-growing cells. The results suggest that the circuit can serve as an effective strain engineering strategy to accelerate biotechnology and molecular biology research.

Next, I constructed another gene circuits, named ADEPT system, to regulate the collective gene expression of an engineered microbial community by modulating plasmid dynamics. By dynamically tuning the plasmid loss rate, horizontal gene transfer rate, and plasmid-mediated fitness effects, I demonstrated that the ADEPT system can tune the total gene expression with a significantly amplified dynamic range.

Finally, in the Appendix, I engineered gene circuits for targeted conjugative plasmid elimination from microbial communities. The results illustrate the potential of plasmid dynamics modulations in engineering complex microbial communities.

https://hdl.handle.net/10161/30797

https://creativecommons.org/licenses/by-nc-nd/4.0/

Biomedical engineering

Systematic biology

Microbiology

Gene circuits

Genetic engineering

Microbiome

Synthetic biology

Programming Microbial Communities via Control of Plasmid Dynamics

Dissertation

24

2026-06-06T13:44:06Z