Natural and synthetic coupling of the Synechococcus elongatus circadian clock

细长聚球藻生物钟的天然和合成耦合

• 批准号: 2119252
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2119252
• 关键词: Natural; synthetic; coupling; Synechococcus; elongatus

Synechococcus elongatus is a model for microbial chronobiology, however, tool limitations leave much to discover about its circadian clock, including definitive answers as to whether they are capable of intercellular coupling. During rotation work, the toolset was expanded, with spectrally distinct fluorescent protein pairings that would allow the dynamics of multiple components to be observed simultaneously, identified. These proteins were built into the S. elongatus laboratory isolate, PCC 7942, as transcriptional reporters under the clock gene promoter, PkaiBC. In current work, spectrally distinct reporter pairs have been used in co-cultures, attempting to empirically determine whether cell-to-cell coupling exists in circadian clocks of S. elongatus. Previous publications suggest an absence of such coupling in PCC 7942, however these studies were subject to limitations. Furthermore, a newly described wild isolate, S. elongatus UTEX 3055, exhibiting many phenotypes lost in PCC 7942, offers the exciting prospect that coupling might also be retained. Initial work thus focused on building equivalent reporters into S. elongatus UTEX 3055. In the process, an adapted protocol for engineering this new isolate has been developed and their circadian dynamics verified. The two fluorescent proteins EYFP-ASV and mTFP1-ASV have been identified as an optimal reporter pairing in both PCC 7942 and UTEX 3055. However, a noticeable phase delay has been observed between the two reporters, thus, efforts have been made to correct for this through phase characterisation experiments.To study potential clock coupling, these EYFP-ASV and mTFP1-ASV reporters in both S. elongatus PCC 7942 and UTEX 3055 backgrounds have been entrained in antiphase and co-cultured, to see whether coupling-induced phase shifts occur. Preliminary data are inconclusive but a process has been developed that improves data quality and, with inclusion of an additional control, this should lead to definitive results. In parallel to studying endogenous coupling, assuming its absence in PCC 7942, work to engineer exogenous coupling mechanisms into the isolate has progressed. Drawing inspiration from the use of quorum-sensing in coupled synthetic biological oscillators, a library of 27 new plasmids has been built, encoding the components from both the Lux and Las quorum-sensing systems from Vibrio fischeri and Pseudomonas aeruginosa respectively. These components target two neutral integration sites in the S. elongatus genome, known as Neutral Site 1 and Neutral Site 2. The engineering of a series of component test strains as well as a set of experiments to fully characterise them has been planned. Information from this testing will ultimately inform the selection of components used in future engineering to connect these quorum-sensing mechanisms to the circadian clock of S. elongatus through a range of genetic network architectures. Characterisation of these genetic components will also help to build mathematical models, which will likewise inform the coupling networks attempted. In doing so, it is hoped that a new type of synthetic oscillator, the behaviour of which is coordinated across a population by the circadian clock, can be built.

细长聚球藻是微生物时间生物学的模型，然而，工具的限制留下了很多关于其生物钟的发现，包括关于它们是否能够进行细胞间耦合的明确答案。在旋转工作中，工具集得到了扩展，确定了光谱上不同的荧光蛋白配对，这将允许同时观察多个组件的动态。这些蛋白质被构建到S.细长体实验室分离物PCC 7942作为时钟基因启动子PkaiBC下的转录报告基因。在目前的工作中，光谱上不同的报告对已被用于共培养，试图凭经验确定是否存在细胞间耦合的昼夜节律钟的S。细长的先前的出版物表明在PCC 7942中不存在这种偶联，然而这些研究受到限制。此外，还发现了一个新的野生分离物S. elongatus UTEX 3055表现出PCC 7942中丢失的许多表型，提供了偶联也可能被保留的令人兴奋的前景。因此，最初的工作集中在将等价的报告基因构建到S。3055.在这个过程中，已经开发了一种用于工程化这种新分离物的适应性方案，并验证了它们的昼夜节律动力学。两种荧光蛋白EYFP-ASV和mTFP 1-ASV已被鉴定为PCC 7942和UTEX 3055中的最佳报告子配对。为了研究潜在的时钟耦合，这些EYFP-ASV和mTFP 1-ASV报告基因在两个S.细长体PCC 7942和UTEX 3055背景的细胞被夹带在反相中并共培养，以观察是否发生偶联诱导的相移。初步数据是不确定的，但已经开发了一个过程，提高数据质量，并包括一个额外的控制，这应该导致明确的结果。在研究内源性偶联的同时，假设PCC 7942中不存在内源性偶联，将外源性偶联机制工程化到分离株中的工作也取得了进展。从耦合合成生物振荡器中使用群体感应中汲取灵感，建立了27个新质粒的文库，分别编码来自费氏弧菌和铜绿假单胞菌的Lux和Las群体感应系统的组分。这些组分靶向S. elongatus基因组，称为中性位点1和中性位点2。已计划对一系列组分测试菌株进行工程设计，并进行一系列实验以充分验证它们。来自该测试的信息将最终告知未来工程中使用的组件的选择，以将这些群体感应机制连接到S. elongatus通过一系列遗传网络架构。这些遗传成分的特征也将有助于建立数学模型，这同样将为所尝试的耦合网络提供信息。在这样做的过程中，人们希望能够建立一种新型的合成振荡器，其行为通过生物钟在整个人群中进行协调。