Targeted control of self-transmissible plasmids by using engineered interfering plasmids

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Targeted control of self-transmissible plasmids by using engineered interfering plasmids Abstract Mobile genetic elements (MGEs) are genetic materials that can move within a genome or between species, a phenomenon known as horizontal gene transfer (HGT). It is well recognized that HGT plays a critical role in introducing, maintaining, and spreading diverse functional traits such as metabolic traits, virulence factors, and antibiotic resistance. For example, in the clinical setting, antibiotic resistance can spread from the resident microflora to invading pathogens or vice versa. Conversely, use of antibiotics can modulate the overall conjugation dynamics by affecting the conjugation efficiency (rate of gene exchange) or by selecting for populations containing mobile plasmids. Therefore, it is critical to develop strategies that can modulate gene persistence by targeting HGT. To this end, we propose to develop a synthetic-biology based intervention strategy that enables targeted suppression or elimination of self-transmissible plasmids. The strategy exploits the vulnerability of conjugation to deliver an engineered plasmid to both suppress the conjugation rate and to accelerate loss of the target plasmid via incompatibility. During conjugation, a mating bridge is established between the donor cell and the recipient cell, allowing one copy of the self-transmissible plasmid to be transferred to the recipient. However, at a smaller efficiency, the transfer apparatus allows a mobilizable (but not self-transmissible) plasmid to be transferred from the recipient to the donor cell. This process is known as retro-transfer. Our design exploits retro-transfer to deliver our engineered plasmid. Upon entry, an incompatibility element carried by our engineered plasmid will expel the self-transmissible plasmid that picks up our plasmid in the first place. This exclusion is enabled by proper control of the selection dynamics. We term this intervention strategy DoS (Denial of Spread) or DDoS (Distributed Denial of Spread), when generalized to the simultaneous targeting of multiple self- transmissible plasmids. Our preliminary modeling and experimental analysis have demonstrated the proof of concept of DoS strategy. Our proposed work will develop and optimize this intervention strategy in depth and apply it to eliminate self-transmissible plasmids encoding antibiotic resistance in pathogenic bacteria. We envision that our proposed work will establish a transformative platform for precise control of gene persistence and flux in microbial communities.