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Development of Molecular Biology and Bioinformatics Tools: From Hydrogen Evolution to Cell Division in Cyanobacteria
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of fossil fuels presents a particularly interesting challenge - our society strongly depends on coal and oil, but we are aware that their use is damaging the environment. Currently, this awareness is gaining momentum, and pressure to evolve towards an energetically cleaner planet is very strong. Molecular hydrogen (H2) is an environmentally suitable energy carrier that could initially supplement or even substitute fossil fuels.

Ideally, the primary energy source to produce hydrogen gas should be renewable, and the process of conversion back to energy without polluting emissions, making this cycle environmentally clean. Photoconversion of water to hydrogen can be achieved using the following strategies: 1) the use of photochemical fuel cells, 2) by applying photovoltaics, or 3) by promoting production of hydrogen by photosynthetic microorganisms, either phototrophic anoxygenic bacteria and cyanobacteria or eukaryotic green algae. For photobiological H2 production cyanobacteria are among the ideal candidates since they: a) are capable of H2 evolution, and b) have simple nutritional requirements - they can grow in air (N2 and CO2), water and mineral salts, with light as the only energy source.

As this project started, a vision and a set of overall goals were established. These postulated that improved H2 production over a long period demanded: 1) selection of strains taking in consideration their specific hydrogen metabolism, 2) genetic modification in order to improve the H2 evolution, and 3) cultivation conditions in bioreactors should be exmined and improved. Within these goals, three main research objectives were set: 1) update and document the use of cyanobacteria for hydrogen production, 2) create tools to improve molecular biology work at the transcription analysis level, and 3) study cell division in cyanobacteria.

This work resulted in: 1) the publication of a review on hydrogen evolution by cyanobacteria, 2) the development of tools to assist understanding of transcription, and 3) the start of a new fundamental research approach to ultimately improve the yield of H2 evolution by cyanobacteria.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2009. , p. 66
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 697
Keywords [en]
Cyanobacteria, hydrogen, RNA, bioinformatics, RT-PCR, RACE, transcription
National Category
Biochemistry and Molecular Biology Bioinformatics and Systems Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-110842ISBN: 978-91-554-7678-6 (print)OAI: oai:DiVA.org:uu-110842DiVA, id: diva2:278532
Public defence
2010-01-20, Häggsalen, Ångströmlaboratoriet, Lägerhyddsv. 1, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-12-21 Created: 2009-11-26 Last updated: 2010-12-22Bibliographically approved
List of papers
1. A brief look at three decades of hydrogen evolution by Cyanobacteria.
Open this publication in new window or tab >>A brief look at three decades of hydrogen evolution by Cyanobacteria.
2002 (English)In: International Journal of Hydrogen Energy, Vol. 27, p. 1209-1215Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-44028 (URN)
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2009-11-13
2. Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria
Open this publication in new window or tab >>Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria
2009 (English)In: BMC Molecular Biology, ISSN 1471-2199, E-ISSN 1471-2199, Vol. 10, p. 79-Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.

RESULTS: With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution.

CONCLUSION: It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-110391 (URN)10.1186/1471-2199-10-79 (DOI)000269645600001 ()19660145 (PubMedID)
Available from: 2009-11-13 Created: 2009-11-13 Last updated: 2017-12-12
3. Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR
Open this publication in new window or tab >>Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR
2006 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 6, p. 31-Article in journal (Refereed) Published
Abstract [en]

Background

In order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed.

Results

The program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA.

Conclusion

The program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample.

Keywords
Tagenerator, RT-PCR
National Category
Biochemistry and Molecular Biology Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:uu:diva-81352 (URN)10.1186/1472-6750-6-31 (DOI)16820068 (PubMedID)
Available from: 2006-08-18 Created: 2006-08-18 Last updated: 2017-12-14Bibliographically approved
4. Webtag: A new web tool providing tags/anchors for RT-PCR experiments with prokaryotes
Open this publication in new window or tab >>Webtag: A new web tool providing tags/anchors for RT-PCR experiments with prokaryotes
2007 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 7, p. 73-Article in journal (Refereed) Published
Abstract [en]

Background: Webtag is a tool providing oligonucleotide sequences (usually called tags or anchors) that are absent from a specified genome. These tags/anchors can be appended to gene specific primers for reverse transcriptase polymerase chain reaction experiments, circumventing genomic DNA contamination. Results: The use of a relational database, in conjunction with a series of scripts written in PHP and Perl, allows the user to rapidly obtain tags that are: 1) suitable for a specific organism, and 2) compatible with other oligonucleotides to be used in the experimental procedures. Conclusion: This new web tool allows scientists to easily and rapidly obtain suitable tags for RTPCR experiments, and is available at http://www.egs.uu.se/software/webtag/.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-13840 (URN)10.1186/1472-6750-7-73 (DOI)000251903100001 ()17961214 (PubMedID)
Available from: 2008-01-28 Created: 2008-01-28 Last updated: 2017-12-11Bibliographically approved
5. A guide for in-house design of template-switch-based 5' rapid amplification of cDNA ends systems
Open this publication in new window or tab >>A guide for in-house design of template-switch-based 5' rapid amplification of cDNA ends systems
2009 (English)In: Analytical biochemistry, ISSN 1096-0309Article in journal (Refereed) Published
Abstract [en]

Rapid amplification of cDNA ends (RACE) is an established strategy used to determine the transcription start point(s) and the 5' untranslated region(s) of mRNA. Different approaches to perform 5' RACE are available, and one particularly simple and powerful strategy is based on a phenomenon called template-switching. We investigated different aspects of template-switch-based 5' RACE, and we describe the different steps leading to the in-house development of a complete 5' RACE system-from oligonucleotide design to polymerase chain reaction (PCR) amplification. We show that the resulting system is reliable, time-efficient, and inexpensive.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-110392 (URN)10.1016/j.ab.2009.10.022 (DOI)000274163900015 ()19837043 (PubMedID)
Available from: 2009-11-13 Created: 2009-11-13 Last updated: 2012-08-22
6. Evidence for transcription of three genes with characteristics of hydrogenases in the green alga Chlamydomonas noctigama
Open this publication in new window or tab >>Evidence for transcription of three genes with characteristics of hydrogenases in the green alga Chlamydomonas noctigama
2010 (English)In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 3, p. 1074-1088Article in journal (Refereed) Published
Abstract [en]

Some green algae have shown the ability to produce hydrogen under anaerobic conditions. The production of hydrogen in green algae is catalyzed by hydrogenases, which are small monomeric enzymes with high conversion efficiency and high oxygen sensitivity. Most green algae analyzed to date where hydrogenase genes are detected, have been shown to contain two distinct hydrogenases. However, very little is known about which functions the two different enzymes represent. There are also many unknowns within the mechanisms behind hydrogen production as to the roles hydrogenases play under different conditions, and consequently also about the potential for optimization of a hydrogen production process which could be found in this respect. This study focuses on the possibility for the presence of more than two hydrogenases in a single green alga. A large number of degenerate primers were designed and used to produce 3′-RACE products, which in turn were used to design gene specific primers used for PCR and 5′-RACE reactions. The sequences were aligned with known algal hydrogenases to identify products which had homology to these. Products where homology was identified were then explored further. A high number of clones from each band were sequenced to identify products with similar lengths which would not show up as separate bands on a gel. Sequences found to have homology with algal hydrogenases were translated into putative amino acid sequences and analyzed further to obtain detailed information about the presence of specific amino acids with known functions in the enzyme. This information was used to evaluate the likelihood of these transcripts coding for true hydrogenases, versus hydrogenase-like or narf-like proteins. We here present evidence showing that Chlamydomonas noctigama is able to transcribe three genes which share a significant number of characteristics with other known algal FeFe-hydrogenases. The three genes have been annotated HYDA1, HYDA2 and HYDA3.

Keywords
Algae, Chlamydomonas noctigama, HYDA, Hydrogen, Hydrogenase
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-110841 (URN)10.1016/j.ijhydene.2009.10.091 (DOI)000274944000023 ()
Available from: 2009-11-26 Created: 2009-11-26 Last updated: 2017-12-12
7. FtsZ in Anabaena sp. PCC 7120; initial characterization of ftsZ transcription and evidence of in vitro FtsZ degradation.
Open this publication in new window or tab >>FtsZ in Anabaena sp. PCC 7120; initial characterization of ftsZ transcription and evidence of in vitro FtsZ degradation.
Show others...
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:uu:diva-110839 (URN)
Available from: 2009-11-26 Created: 2009-11-26 Last updated: 2009-11-27

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