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

利用工程干扰质粒靶向控制自传载体 摘要 可移动遗传元件(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起着至关重要的作用 在引入、维持和传播不同的功能性状方面的作用，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传播 微生物区系对入侵的病原体或反之亦然。相反，抗生素的使用可以调节整体 通过影响接合效率(基因交换率)或通过选择 含有流动质粒的种群。因此，开发能够调节基因的策略是至关重要的 以HGT为目标的坚持。 为此，我们建议开发一种基于合成生物学的干预策略，使 有针对性地抑制或消除可自我传播的质粒。该策略利用了该漏洞 通过接合传递工程质粒来抑制接合率和加速接合率损失 通过不相容的方式获得目的质粒。在接合过程中，供体之间建立了一座交配桥梁 细胞和受体细胞，允许一份自我传播的质粒被转移到受体。 然而，以较小的效率，转移装置允许可动员的(但不能自我传播的)质粒 从受者细胞转移到供者细胞。此处理称为追溯调动。我们的设计充分利用了 逆转录-转移，以传递我们的工程质粒组。一旦进入，由我们的工程人员携带的不相容元素 首先，质粒会排出携带我们的质粒的自我传播的质粒。这一排除是 通过对选择动态的适当控制来实现。我们称这种干预策略为DoS(拒绝传播) 或DDoS(分布式拒绝传播)，当推广到同时瞄准多个自我 可传播的质粒。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耐药性的自我传播质粒。我们 设想我们提议的工作将为精确控制基因持久性建立一个变革性的平台 和微生物群落中的通量。

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.