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Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Mikrobiell kemi, Microbial chemistry)
Bioforsk, Norge.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Mikrobiell kemi, Microbial chemistry)
2014 (English)In: Journal of Biological Engineering, ISSN 1754-1611, Vol. 8, no 4Article in journal (Refereed) Published
Abstract [en]


Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from Escherichia coli is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary lac operator (lacO), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated lacO and loops the DNA. Ptrc is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium Synechocystis PCC 6803. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of Ptrc and cyanobacterial DNA-looping, we designed a Ptrc-derived promoter library consisting of single lacO promoters, including a version of Ptrc with a stronger lacO (Ptrc1O-proximal), and dual lacO promoters with varying inter-lacO distances (the Ptrc2O-library).


We first characterized artificial constitutive promoters and used one for engineering a LacI-expressing strain of Synechocystis. Using this strain, we observed that Ptrc1O-proximal is similar to Ptrc in being inefficiently repressed. Further, the Ptrc2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter-lacO distances, in both E. coli and Synechocystis. Repression of Ptrc2O-library promoters with operators out of phase is less efficient in Synechocystis than in E. coli, whereas repression of promoters with lacO in phase is efficient even under induced conditions in Synechocystis. Two well-repressed Ptrc2O promoters were highly active when tested in absence of LacI in Synechocystis.


The artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of Ptrc and Ptrc1O-proximal in Synechocystis, as compared to E. coli, may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in E. coli. DNA-looping contributes strongly to Ptrc2O-library repression in Synechocystis, even though they contain the weakly-repressed primary lacO of Ptrc1O-proximal and relatively low levels of LacI/cell. Hence, Synechocystis RNAP may be more sensitive to DNA-looping than E. coli RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed Ptrc2O promoters could be used without induction, or together with an unstable LacI.

Place, publisher, year, edition, pages
2014. Vol. 8, no 4
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-217339DOI: 10.1186/1754-1611-8-4ISI: 000335793400001OAI: oai:DiVA.org:uu-217339DiVA: diva2:692902
Available from: 2014-02-03 Created: 2014-02-03 Last updated: 2014-06-30Bibliographically approved
In thesis
1. 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1149
Cyanobacteria, Synthetic biology, promoters, transcription, LacI, Gal4, Light-regulation
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry with specialization in Microbial Chemistry
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)
Available from: 2014-05-15 Created: 2014-04-22 Last updated: 2014-06-30

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