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Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
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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.

Place, publisher, year, edition, pages
2011. Vol. 497, 539-579 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-155689DOI: 10.1016/B978-0-12-385075-1.00024-XISI: 000291321200024OAI: oai:DiVA.org:uu-155689DiVA: diva2:428367
Available from: 2011-06-30 Created: 2011-06-28 Last updated: 2017-12-11
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1063
Keyword
synthetic biology, cyanobacteria, promoter, engineering, TetR, DNA breathing dynamics, transcription, regulation
National Category
Biochemistry and Molecular Biology
Identifiers
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)
Opponent
Supervisors
Available from: 2013-09-06 Created: 2013-09-06 Last updated: 2015-07-29Bibliographically approved
2. Engineering Transcriptional Systems for Cyanobacterial Biotechnology
Open this publication in new window or tab >>Engineering Transcriptional Systems for Cyanobacterial Biotechnology
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyanobacteria are solar-powered cell factories that can be engineered to supply us with renewable fuels and chemicals. To do so robust and well-working biological parts and tools are necessary. Parts for controlling gene expression are of special importance in living systems, and specifically promoters are needed for enabling and simplifying rational design. Synthetic biology is an engineering science that incorporates principles such as decoupling, standardization and modularity to enable the design and construction of more advanced systems from simpler parts and the re-use of parts in new contexts. For these principles to work, cross-talk must be avoided and therefore orthogonal parts and systems are important as they are decoupled by definition. This work concerns the design and development of biological parts and tools that can enable synthetic biology in cyanobacteria. This encompasses parts necessary for the development of other systems, such as vectors and translational elements, but with a focus on transcriptional regulation. First, to enable the development and characterization of promoters in different cyanobacterial chassis, a broad-host-range BioBrick plasmid, pPMQAK1, was constructed and confirmed to function in several cyanobacterial strains. Then, ribosome binding sites, protease degradation tags and constitutive, orthogonal promoters were characterized in the model strain Synechocystis PCC 6803. These tools were then used to design LacI-regulated promoter libraries for studying DNA-looping and the behaviour of LacI-mediated loops in Synechocystis. Ultimately, this lead to the design of completely repressed LacI-regulated promoters that could be used for e.g. cyanobacterial genetic switches, and was used to design a destabilized version of the repressed promoter that could be induced to higher levels. Further, this promoter was used to implement an orthogonal transcriptional system based on T7 RNAP that was shown to drive different levels of T7 promoter transcription depending on regulation. Also, Gal4-repressed promoters for bacteria were engineered and examined in Escherichia coli as an initial step towards transferring them to cyanobacteria. Attempts were also made to implement a light-regulated one-component transcription factor based on Gal4. This work provides a background for engineering transcription and provides suggestions for how to develop the parts further.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 63 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1149
Keyword
Cyanobacteria, Synthetic biology, promoters, transcription, LacI, Gal4, Light-regulation
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry with specialization in Microbial Chemistry
Identifiers
urn:nbn:se:uu:diva-223599 (URN)978-91-554-8954-0 (ISBN)
Public defence
2014-06-05, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-05-15 Created: 2014-04-22 Last updated: 2014-06-30

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Heidorn, ThorstenCamsund, DanielHuang, Hsin-HoLindberg, PiaOliveira, PauloStensjö, KarinLindblad, Peter

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