Natural and synthetic coupling of the Synechococcus elongatus circadian clock

细长聚球藻是微生物时间生物学的一个模型，然而，工具的限制留下了许多关于它的生物钟的发现，包括它们是否能够进行细胞间耦合的确切答案。在轮换工作期间，工具箱得到了扩展，使用了光谱不同的荧光蛋白质对，这将允许同时观察和识别多个组分的动态。这些蛋白被构建到长链霉菌实验室分离株PCC7942中，作为转录报告基因在时钟基因启动子PkaiBC的作用下。在目前的工作中，光谱不同的报告对已经被用于共培养，试图经验性地确定在细长链霉菌的昼夜节律中是否存在细胞到细胞的耦合。以前的研究表明，在PCC7942中没有这种关联，然而这些研究受到限制。此外，一个新描述的野生分离株，长链链球菌UTEX 3055，表现出许多表型在PCC 7942中丢失，提供了令人兴奋的前景，耦合也可能被保留。因此，最初的工作集中在将等同的记者建立在拉长链霉菌UTEX 3055中。在这个过程中，已经开发了一种适合于设计这种新分离株的方案，并验证了它们的昼夜节律动力学。两种荧光蛋白EYFP-ASV和mTFP1-ASV已被确定为PCC 7942和UTEX 3055中的最佳报告配对。为了研究潜在的时钟耦合，在细长链霉菌PCC 7942和UTEX 3055背景中的这些EYFP-ASV和mTFP1-ASV报告器被携带在反相和共培养中，以观察是否发生耦合诱导的相移。初步数据尚无定论，但已开发出一种提高数据质量的程序，加上额外的对照，应可得出确定的结果。在研究内源性耦合的同时，假设在PCC7942中没有内源性耦合，将外源耦合机制工程到隔离中的工作已经取得进展。受群体感应在耦合合成生物振荡器中使用的启发，已经建立了一个由27个新的质粒库组成的文库，分别编码来自鱼弧菌和铜绿假单胞菌的Lux和LAS群体感应系统的成分。这些成分针对长链霉菌基因组中的两个中性整合位点，即中性位点1和中性位点2。一系列成分测试菌株的工程以及一系列实验已经计划好，以充分描述它们的特征。来自这项测试的信息最终将为未来工程中使用的组件的选择提供信息，以通过一系列遗传网络结构将这些群体感应机制连接到细长链霉菌的生物钟。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，人们希望能够建造一种新型的合成振荡器，其行为通过生物钟在整个种群中进行协调。

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.