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Promoter Engineering for Cyanobacteria: An Essential Step
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
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
Keyword [en]
synthetic biology, cyanobacteria, promoter, engineering, TetR, DNA breathing dynamics, transcription, regulation
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-206901ISBN: 978-91-554-8724-9OAI: oai:DiVA.org:uu-206901DiVA: diva2:646065
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
List of papers
1. Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology
Open this publication in new window or tab >>Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology
2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 8, 2577-2593 p.Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria are suitable for sustainable, solar-powered biotechnological applications. Synthetic biology connects biology with computational design and an engineering perspective, but requires efficient tools and information about the function of biological parts and systems. To enable the development of cyanobacterial Synthetic Biology, several molecular tools were developed and characterized: (i) a broad-host-range BioBrick shuttle vector, pPMQAK1, was constructed and confirmed to replicate in Escherichia coli and three different cyanobacterial strains. (ii) The fluorescent proteins Cerulean, GFPmut3B and EYFP have been demonstrated to work as reporter proteins in cyanobacteria, in spite of the strong background of photosynthetic pigments. (iii) Several promoters, like P-rnpB and variants of P-rbcL, and a version of the promoter P-trc with two operators for enhanced repression, were developed and characterized in Synechocystis sp. strain PCC6803. (iv) It was shown that a system for targeted protein degradation, which is needed to enable dynamic expression studies, is working in Synechocystis sp. strain PCC6803. The pPMQAK1 shuttle vector allows the use of the growing numbers of BioBrick parts in many prokaryotes, and the other tools herein implemented facilitate the development of new parts and systems in cyanobacteria.

National Category
Chemical Sciences
urn:nbn:se:uu:diva-137784 (URN)10.1093/nar/gkq164 (DOI)000277238900008 ()
Available from: 2010-12-15 Created: 2010-12-15 Last updated: 2014-06-30
2. Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions
Open this publication in new window or tab >>Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions
Show others...
2011 (English)In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 497, 539-579 p.Article, review/survey (Refereed) Published
Abstract [en]

Cyanobacteria are the only prokaryotes capable of using sunlight as their energy, water as an electron donor, and air as a source of carbon and, for some nitrogen-fixing strains, nitrogen. Compared to algae and plants, cyanobacteria are much easier to genetically engineer, and many of the standard biological parts available for Synthetic Biology applications in Escherichia coli can also be used in cyanobacteria. However, characterization of such parts in cyanobacteria reveals differences in performance when compared to E. coli, emphasizing the importance of detailed characterization in the cellular context of a biological chassis. Furthermore, cyanobacteria possess special characteristics (e.g., multiple copies of their chromosomes, high content of photosynthetically active proteins in the thylakoids, the presence of exopolysaccharides and extracellular glycolipids, and the existence of a circadian rhythm) that have to be taken into account when genetically engineering them. With this chapter, the synthetic biologist is given an overview of existing biological parts, tools and protocols for the genetic engineering, and molecular analysis of cyanobacteria for Synthetic Biology applications.

National Category
Natural Sciences
urn:nbn:se:uu:diva-155689 (URN)10.1016/B978-0-12-385075-1.00024-X (DOI)000291321200024 ()
Available from: 2011-06-30 Created: 2011-06-28 Last updated: 2016-04-21
3. Wide-dynamic-range promoters engineered for cyanobacteria
Open this publication in new window or tab >>Wide-dynamic-range promoters engineered for cyanobacteria
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.

National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-205305 (URN)10.1186/1754-1611-7-10 (DOI)000334096700009 ()23607865 (PubMedID)
EU, European Research Council, 308518
Available from: 2013-08-15 Created: 2013-08-15 Last updated: 2014-05-26Bibliographically approved
4. A point mutation downstream of the -10 promoter element does not exhibit long-range effect on TetR binding
Open this publication in new window or tab >>A point mutation downstream of the -10 promoter element does not exhibit long-range effect on TetR binding
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-205306 (URN)
Available from: 2013-08-15 Created: 2013-08-15 Last updated: 2015-06-22

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