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

利用工程干扰质粒靶向控制自身传播质粒 摘要 移动的遗传元件（MGE）是可以在基因组内或基因组之间移动的遗传物质。 这种现象被称为水平基因转移（HGT）。众所周知，HGT发挥着关键的作用， 在引入、维持和传播多种功能性状如代谢性状、毒力因子、 和抗生素耐药性。例如，在临床环境中，抗生素耐药性可以从居民 微生物菌群与入侵的病原体或反之亦然。相反，使用抗生素可以调节整体 通过影响偶联效率（基因交换速率）或通过选择 含有移动的质粒的群体。因此，开发能够调控基因表达的策略至关重要。 坚持以HGT为目标。 为此，我们建议开发一种基于合成生物学的干预策略， 靶向抑制或消除自身传播质粒。该策略利用了 以递送工程化质粒，从而抑制接合速率并加速缀合物的损失。 通过不相容性将目标质粒分离。在接合过程中，在供体之间建立了一个配对桥， 细胞和受体细胞，允许将一个拷贝的自传递质粒转移至受体。 然而，在较小的效率下，转移装置允许可移动的（但不是自传递的）质粒 从受体转移到供体细胞。这个过程被称为逆向转移。我们的设计利用了 逆转录转移来传递我们的工程质粒。进入后，我们的工程师携带的不相容元素 质粒会排出携带我们质粒的自我传播质粒。这种排斥是 通过选择动力学的适当控制来实现。我们将这种干预策略称为DoS（拒绝传播） 或DDoS（分布式拒绝传播），当推广到多个自我的同时目标， 可传递质粒我们的初步建模和实验分析证明了 DoS策略的概念。我们建议的工作将深入发展和优化这一干预战略， 将其应用于消除病原菌中编码抗生素抗性的自我传播质粒。我们 我认为我们的工作将为精确控制基因持久性建立一个变革性平台 和微生物群落的流动。

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.