Natural and synthetic coupling of the Synechococcus elongatus circadian clock

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.