TetR-regulated Promoter Library Showing Wide Dynamic Range in the Cyanobacterium Synechocystis PCC 6803

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Hsin-Ho Huang, Peter Lindblad

Uppsala University, Sweden

The functional promoter library regulated by the TetR repressor was demonstrated with the reporter EYFP (enhanced yellow fluorescent protein) gene, for the first time, in the unicellular cyanobacterium Synechocystis sp. strain ATCC27184 (i.e. glucose-tolerant Synechocystis sp. strain PCC 6803). Some promoters in the library show appreciably wide dynamic range of transcription regulation; about 200-fold gene induction in 24 or 48 hours depending on the growth condition. The key strategy of promoter engineering is to alter the transcription initiation by systematic changes of few nucleotides in the region between the -10 element and the transcription start site. This region coincides with the “discriminator” region in the case of rRNA or tRNA promoter of Escherichia coli, which has been shown its effective role in affecting transcription initiation [1]. Thanks to the versatile properties of well-developed TetR repressor regulation system [2], the present promoter library can contribute to implement novel functionalities in the chassis [3] derived from this photosynthetic microorganism via layering new genetic circuits [4] in the framework of synthetic biology. REFERENCES 1. Haugen SP, Berkmen MB, Ross W, Gaal T, Ward C, Gourse RL: rRNA promoter regulation by nonoptimal binding of sigma region 1.2: an additional recognition element for RNA polymerase. Cell 2006, 125:1069-1082. 2. Bertram R, Hillen W: The application of Tet repressor in prokaryotic gene regulation and expression. Microb Biotechnol 2008, 1:2-16. 3. Pinto F, van Elburg KA, Pacheco CC, Lopo M, Noirel J, Montagud A, Urchueguia JF, Wright PC, Tamagnini P: Construction of a chassis for hydrogen production: physiological and molecular characterization of a Synechocystis sp. PCC 6803 mutant lacking a functional bidirectional hydrogenase. Microbiology 2012, 158:448-464. 4. Moon TS, Lou C, Tamsir A, Stanton BC, Voigt CA: Genetic programs constructed from layered logic gates in single cells. Nature 2012.