Natural and synthetic coupling of the Synechococcus elongatus circadian clock

长聚球菌是微生物时间生物学的一个模型，然而，工具的限制留下了很多关于其生物钟的发现，包括它们是否能够细胞间偶联的明确答案。在轮换工作期间，工具集得到了扩展，具有光谱上不同的荧光蛋白配对，可以同时观察和识别多个组分的动态。这些蛋白被构建到S. elongatus实验室分离物PCC 7942中，作为时钟基因启动子PkaiBC下的转录报告蛋白。在目前的工作中，在共培养中使用了光谱上不同的报告基因对，试图以经验确定长形棘球蚴的生物钟中是否存在细胞间耦合。先前的出版物表明在PCC 7942中缺乏这种耦合，然而这些研究受到限制。此外，新发现的野生分离株S. elongatus UTEX 3055显示出PCC 7942中丢失的许多表型，这为偶联可能保留提供了令人兴奋的前景。因此，最初的工作集中在将等效报告器构建到S. elongatus UTEX 3055中。在此过程中，开发了一种新的工程方案，并验证了它们的昼夜动力学。两个荧光蛋白EYFP-ASV和mTFP1-ASV已被确定为PCC 7942和UTEX 3055的最佳报告基因配对。然而，在两个报告者之间观察到明显的相位延迟，因此，已经努力通过相位表征实验来纠正这一点。为了研究潜在的时钟耦合，我们将长形海螺PCC 7942和UTEX 3055背景下的EYFP-ASV和mTFP1-ASV报告基因置于反相和共培养中，以观察耦合诱导的相移是否发生。初步数据尚无定论，但已制定了一项改进数据质量的程序，并包括一项额外的控制措施，这将导致确定的结果。在研究内源性偶联的同时，假设PCC 7942中不存在外源性偶联机制，在分离物中设计外源性偶联机制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的应用启发，我们建立了一个包含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。这些成分靶向长尾螺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。已经计划了一系列组件测试菌株的工程以及一套充分表征它们的实验。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，这些组件将通过一系列遗传网络结构将这些群体感应机制与S. elongatus的生物钟连接起来。这些遗传成分的特征也将有助于建立数学模型，这同样将告知耦合网络的尝试。在此过程中，人们希望能够制造出一种新型的合成振荡器，这种振荡器的行为可以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.