Cyanobacteria are unique among prokaryotes in that they can conduct oxygenic photosynthesis. With just the addition of light, water and some trace minerals, cyanobacteria utilise carbon dioxide to synthesise the simple carbohydrates required to produce the chemical energy that drives all cellular processes. Cyanobacteria have a complex metabolism when compared to other model heterotrophs (e.g. Escherichia coli) and therefore can produce a wide variety of complex biomolecules not possible in other prokaryotes. Although cyanobacteria show great potential for green biotechnology applications, availability of molecular tools and strategies required to drive forward basic research and the engineering of new strains alike, has been quite limited.
To address the lack of a unified strategy for the engineering of cyanobacteria, we developed a molecular cloning system called CyanoGate that unifies cyanobacteria and plants. This system is based on the widely adopted modular and high throughput Golden Gate cloning syntax. CyanoGate contains a suite of well characterised modular parts and acceptors for episomal and chromosomal gene expression, genome engineering applications, and CRISPR interference and sRNA tools for gene repression studies.
Building on the CyanoGate platform, I adapted a strategy for the evaluation of transcription terminators. Transcription terminators are important control elements for the regulation of gene expression, and there have been relatively few studies limited only to the model species Synechocystis sp. PCC 6803 thus far. Here, I have constructed and validated a high throughput molecular tool that can be used in any organism where the broad host range RSF1010 origin of replication is functional. With this tool, a library of transcription terminators was characterised and compared between Escherichia coli, Synechocystis sp. PCC 6803 and Synechococcus elongatus UTEX 2973. Surprisingly, our findings showed that transcription termination efficiency was not only different between E. coli and cyanobacteria, but the library also performed differently between cyanobacterial species.
Lastly, I investigated several heterologous inducible and repressible expression systems in Synechocystis. I developed a rhamnose-responsive genetic inverter with a range of output strengths using a transcription factor repressor new to cyanobacteria. There are very few inducible and repressible systems thus far reported as functional in cyanobacteria, and this new repressor will be a useful addition for the construction of more complex gene circuits in cyanobacteria
