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Wide-dynamic-range promoters engineered for cyanobacteria
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
2013 (English)In: Journal of Biological Engineering, ISSN 1754-1611, Vol. 7, no 1, 10- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Cyanobacteria, prokaryotic cells with oxygenic photosynthesis, are excellent bioengineering targets to convert solar energy into solar fuels. Tremendous genetic engineering approaches and tools have been and still are being developed for prokaryotes. However, the progress for cyanobacteria is far behind with a specific lack of non-native inducible promoters.

RESULTS: We report the development of engineered TetR-regulated promoters with a wide dynamic range of transcriptional regulation. An optimal 239 (±16) fold induction in darkness (white-light-activated heterotrophic growth, 24 h) and an optimal 290 (±93) fold induction in red light (photoautotrophic growth, 48 h) were observed with the L03 promoter in cells of the unicellular cyanobacterium Synechocystis sp. strain ATCC27184 (i.e. glucose-tolerant Synechocystis sp. strain PCC 6803). By altering only few bases of the promoter in the narrow region between the -10 element and transcription start site significant changes in the promoter strengths, and consequently in the range of regulations, were observed.

CONCLUSIONS: The non-native inducible promoters developed in the present study are ready to be used to further explore the notion of custom designed cyanobacterial cells in the complementary frameworks of metabolic engineering and synthetic biology.

Place, publisher, year, edition, pages
2013. Vol. 7, no 1, 10- p.
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-205305DOI: 10.1186/1754-1611-7-10ISI: 000334096700009PubMedID: 23607865OAI: oai:DiVA.org:uu-205305DiVA: diva2:641188
EU, European Research Council, 308518
Available from: 2013-08-15 Created: 2013-08-15 Last updated: 2014-05-26Bibliographically approved
In thesis
1. Promoter Engineering for Cyanobacteria: An Essential Step
Open this publication in new window or tab >>Promoter Engineering for Cyanobacteria: An Essential Step
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Synthetic biology views a complex biological system as an ensemble in the hierarchy of parts, devices, systems, and networks. The practice of using engineering rules such as decoupling and standardization to understand, predict, and re-build novel biological functions from model-driven designed genetic circuits is emphasized. It is one of the top ten technologies that could help solving the current and potential risks in human society. Cyanobacteria have been considered as a promising biological system in conducting oxygenic photosynthesis to convert solar energy into reducing power, which drives biochemical reactions to assimilate and generate chemicals for a specific purpose such as CO2 fixation, N2 fixation, bioremediation, or fuels production. The promoter is a key biological part to construct feedback loops in genetic circuits for a desired biological function. In this thesis, promoters that don't work in the cyanobacterium Synechocystis PCC 6803 in terms of promoter strength, and dynamic range of gene regulation are identified. Biological parts, such as ribosome binding sites, and reporter genes with and without protease tags were also characterized with the home-built broad-host-range BioBrick shuttle vector pPMQAK1. The strong L03 promoter, which can be tightly regulated in a wide dynamic range by the foreign Tet repressor, was created through an iterative promoter engineering cycle. The iteration cycle of DNA breathing dynamic simulations and quantification of a reporting signal at a single-cell level should guide through the engineering process of making promoters with intended regulatory properties. This thesis is an essential step in creating functional promoters and it could be applied to create more diverse promoters to realize the emphasized practices of synthetic biology to build synthetic cyanobacteria for direct fuel production and CO2 assimilation.

Place, publisher, year, edition, pages
Acta Universitatis Upsaliensis, 2013. 58 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1063
synthetic biology, cyanobacteria, promoter, engineering, TetR, DNA breathing dynamics, transcription, regulation
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-206901 (URN)978-91-554-8724-9 (ISBN)
Public defence
2013-09-27, Polacksbacken, Pol_2146, Lägerhyddsvägen 1, Uppsala, 13:15 (English)
Available from: 2013-09-06 Created: 2013-09-06 Last updated: 2015-07-29Bibliographically approved

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