Development of Artificial Transcriptional Control Systems for Cyanobacteria

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Daniel Camsund, Thorsten Heidorn, Alfonso Jaramillo, Peter Lindblad

Uppsala University, Sweden

Cyanobacteria are photosynthetic prokaryotes of great biotechnological potential as they can use sunlight as energy source and CO2 from air as carbon source. Engineering these solar-powered bacteria to produce biofuels could supply us with the renewable fuels of the future. However, to facilitate this development, robust and versatile biological parts and tools for engineering cyanobacteria are needed. Among these tools, the control of gene expression is crucial as it is the first step in expressing any functional element inside a living cell. Thus, we develop transcriptional control systems for use in cyanobacteria. The first is a LacI-regulated system based on a modified version of the strong artificial trc promoter that contains a single lac operator. We add another lacO and vary its position to investigate how this affects DNA-looping and transcriptional regulation in both Escherichia coli and the cyanobacterium Synechocystis sp. PCC 6803. This information is important in identifying differences between the native transcriptional machineries of E. coli and Synechocystis, facilitating the design of an optimal LacI-regulated promoter. A second system relies on light for regulation of gene expression. Light is an excellent regulator of gene expression as it is transient and non-invasive, easy to integrate in different systems, and has been demonstrated for in vivo gene regulation using fully genetically encoded light sensors. Furthermore, as sunshine is the energy source of cyanobacteria, being able to synchronize genetic circuits with day/night cycles through a light-regulated orthogonal gene expression system could provide advantages in solar-powered biotechnology. To achieve this we develop light-regulated transcriptional systems that control promoters directly through the binding of light-sensitive dimers of transcription factors.