Investigation of natural and synthetic coupling of the circadian clock in the cyanobacterium Synechococcus elongatus

*Synechococcus* *elongatus*, owing to its possession of a simple, yet robust circadian clock, is a well-studied model for microbial chronobiology. However, historical tool limitations have left much still to learn about this clock, including definitive answers as to whether it exhibits coupling. During this PhD project, the tool set has been expanded and subsequently applied to the study of clock coupling, in an attempt to provide such answers. First, to enable multiplexed observation of gene expression dynamics, spectrally distinct fluorescent protein reporter pairings were identified. A shortlist of such proteins: mTFP1, mTQ2, EYFP and mNG, were then built as transcriptional reporters of the P*kaiBC* circadian promoter, into both the *S.* *elongatus* laboratory isolate, PCC 7942 and the recently described wild isolate, UTEX 3055. UTEX 3055 is noteworthy for retaining several phenotypes lost in PCC 7942, including those associated with intracellular coordination, such as biofilm formation, offering the exciting prospect that clock coupling might also be retained. Characterisation of these tools in PCC 7942 and UTEX 3055 confirmed faithful reporting on clock status, revealing highly similar circadian dynamics in both isolates. Furthermore, an optimal reporter pairing of mTFP1 and EYFP was identified, which was verified to be appropriate for multiplexed observation of clock populations in co-culture. Armed with these new tools for both PCC 7942 and UTEX 3055, the opportunity was taken to investigate clock coupling more directly than was previously possible: via differentially entrained spectrally distinct reporter co-cultures. While liquid- and solid-state co-cultures were attempted, entrained in both antiphase and an intermediate phase difference, no evidence for coupling was found in either isolate. Simultaneously, inspired by shared principles between synthetic biological and circadian oscillators, efforts were also made to expand the synthetic biological toolbox for oscillator coupling. Emphasis was placed on developing coupling tools derived from Gram-negative bacterial quorum-sensing systems, which have been used to great effect in coordinating synthetic microbial consortia. Their implementation in *S.* *elongatus* as synthetic clock-coupling mechanisms may yield both simple model systems for studying clock coupling and circadian control systems for biotechnological applications. To this end, engineering focused on introducing components from 19 diverse quorum-sensing systems to *S.* *elongatus* PCC 7942: the Ahy system from *Aeromonas* *hydrophilia*; the Aub system from a hitherto unidentified *uncultured* *bacterium*; the Bja system from *Bradyrhizobium* *diazoefficiens*; the Bra system from *Paraburkholderia* *kururiensis*; the Cep system from *Burkholderia* *cepacia*; the Cer system from *Rhodobacter* *sphaeroides*; the Cin system from *Rhizobium* *leguminosarum*; the Esa system from *Pantoea* *stewartii*; the Exp system from *Erwinia* *chrysanthemi*; the Las system from *Pseudomonas* *aeruginosa*; the Lux system from *Vibrio* *fischeri*; the Rhl system from *Pseudomonas* *aeruginosa*; the Rpa system from *Rhodopseudomonas* *palustris*; the Sin system from *Sinorhizobium* *meliloti*; the Sma system from *Serratia* sp. ATCC 39006; the Spl system from *Serratia* *plymuthica*; the Spn system from *Serratia* *marascens*; the Spr system from *Serratia* *proteomaculans*; and the Tra system from *Agrobacterium* *tumefaciens*. Owing to their standalone utility as new inducible promoter systems for *S.* *elongatus*, priority was placed on building homoserine lactone-inducible “receiver” modules, for which strain engineering was completed for 15 out of the 19 systems at the time of writing. Additionally, “sender” strains were also built for two of the best-characterised quorum-sensing systems in synthetic biology, the Lux and Las quorum-sensing systems. Due to time constraints, only characterisation of the Lux quorum-sensing system had been attempted in time to be included in this thesis. Although Lux receiver results were promising, exhibiting response to exogenously applied cognate HSL inducer, no response was seen when co-cultured with sender strains. However, receivers also exhibited a response to non-cognate HSL inducer belonging to the Las system, thus, the possibility that Lux receivers might be induced when co-cultured with Las senders remains a possibility. Prospects will be further enhanced once construction and characterisation of all remaining quorum-sensing receivers is complete. This will constitute an extensive library of new inducible modules for synthetic biology, representing appreciable progress towards eventual implementation of synthetic coupling in the species.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Micklem, Christopher

Advisor dc:contributor.advisor

• Locke, James

• Circadian clock
• Oscillator
• Coupling
• Cyanobacteria
• Quorum sensing
• Chronobiology
• Synthetic biology
• Engineering biology
• Systems biology

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1a565de1-19c1-4dda-b391-e7d4b0735904/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0001-9475-5559

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/376562