Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Engineering Transcriptional Systems for Cyanobacterial Biotechnology
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. (Mikrobiell kemi)
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 [en]
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: urn:nbn:se:uu:diva-223599ISBN: 978-91-554-8954-0 (print)OAI: oai:DiVA.org:uu-223599DiVA: diva2:713315
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
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
Identifiers
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
Identifiers
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. Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium
Open this publication in new window or tab >>Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium
2014 (English)In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 8, no 4Article in journal (Refereed) Published
Abstract [en]

Background

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).

Results

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.

Conclusions

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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-217339 (URN)10.1186/1754-1611-8-4 (DOI)000335793400001 ()
Available from: 2014-02-03 Created: 2014-02-03 Last updated: 2017-12-06Bibliographically approved
4. A LacI-regulated promoter for Synechocystis and its use for implementing a T7 RNA polymerase-based orthogonal transcriptional system
Open this publication in new window or tab >>A LacI-regulated promoter for Synechocystis and its use for implementing a T7 RNA polymerase-based orthogonal transcriptional system
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-223598 (URN)
Available from: 2014-04-22 Created: 2014-04-22 Last updated: 2014-06-30
5. Genetically engineered light sensors for control of bacterial gene expression
Open this publication in new window or tab >>Genetically engineered light sensors for control of bacterial gene expression
2011 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, no 7, 826-836 p.Article, review/survey (Refereed) Published
Abstract [en]

Light of different wavelengths can serve as a transient, noninvasive means of regulating gene expression for biotechnological purposes. Implementation of advanced gene regulatory circuits will require orthogonal transcriptional systems that can be simultaneously controlled and that can produce several different control states. Fully genetically encoded light sensors take advantage of the favorable characteristics of light, do not need the supplementation of any chemical inducers or co-factors, and have been demonstrated to control gene expression in Escherichia coli. Herein, we review engineered light-sensor systems with potential for in vivo regulation of gene expression in bacteria, and highlight different means of extending the range of available light input and transcriptional output signals. Furthermore, we discuss advances in multiplexing different light sensors for achieving multichromatic control of gene expression and indicate developments that could facilitate the construction of efficient systems for light-regulated, multistate control of gene expression.

Keyword
Multichromatic, light-regulated promoter, Photoreceptor, Protein engineering, Synthetic biology
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-156829 (URN)10.1002/biot.201100091 (DOI)000292738100007 ()
Available from: 2011-08-11 Created: 2011-08-09 Last updated: 2017-12-08Bibliographically approved
6. Development of Gal4-regulated transcriptional systems in Escherichia coli
Open this publication in new window or tab >>Development of Gal4-regulated transcriptional systems in Escherichia coli
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-223565 (URN)
Available from: 2014-04-22 Created: 2014-04-22 Last updated: 2014-06-30

Open Access in DiVA

fulltext(10980 kB)2300 downloads
File information
File name FULLTEXT01.pdfFile size 10980 kBChecksum SHA-512
0612042c221dea1726223f03e80a773f776e05aa1dd98a0e8bafa17c2b23f14ebe8124b1d6780b5e20059e92ba4a078fb4db98cbfb1a295f54a1e4ae505bc84f
Type fulltextMimetype application/pdf
Buy this publication >>

Authority records BETA

Camsund, Daniel

Search in DiVA

By author/editor
Camsund, Daniel
By organisation
Molecular Biomimetics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 2300 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 2744 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf