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Utilising the native plasmid, pCA2.4, from the cyanobacterium synechocystis sp. strain PCC6803 as a cloning site for enhanced product production

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Show simple item record Armshaw, Patricia Carey, Dawn Pembroke, Tony J. 2016-02-25T17:02:23Z 2016-02-25T17:02:23Z 2015
dc.identifier.citation Patricia Armshaw, Dawn Carey, Con Sheahan and J. Tony Pembroke (2015) 'Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced product production'. Biotechnology for Biofuels, 8 (201). en_US
dc.description peer-reviewed en_US
dc.description.abstract BackgroundThe use of photosynthetic autotrophs and in particular the model organism Synechocystis PCC6803 is receiving much attention for the production of sustainable biofuels and other economically useful products through metabolic engineering. Optimisation of metabolic-engineered organisms for high-level sustained production of product is a key element in the manipulation of this organism. A limitation to the utilisation of metabolically-engineered Synechocystis PCC6803 is the availability of strong controllable promoters and stable gene dosage methods for maximising gene expression and subsequent product formation following genetic manipulation.ResultsA native Synechocystis PCC6803 small plasmid, pCA2.4, is consistently maintained at a copy level of up to 7 times that of the polyploid chromosome. As this plasmid is stable during cell division, it is potentially an ideal candidate for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expression vector generated from the native plasmid, pCA2.4. To investigate the feasibility of this new expression system, a yellow fluorescent protein (YFP) encoding gene was cloned downstream of the strong Ptrc promoter and integrated into a predicted neutral site within the pCA2.4 plasmid. The stability of the integrated construct was monitored over time compared to a control strain containing an identical YFP-expressing construct integrated at a known neutral site in a chromosomal location.ConclusionsA significantly higher fluorescence level of the yellow fluorescent protein was observed when its encoded gene was integrated into the pCA2.4 native plasmid when compared to the isogenic chromosomally integrated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integration into the native plasmid. Fluorescence was also monitored as a function of culture time and demonstrated to be stable over multiple sub-cultures even after the removal of selective pressure. Therefore, the native small plasmid, pCA2.4 may be utilised to stably increase gene expression levels in Synechocystis PCC6803. With the complementary utilisation of an inducible promoter system, rapid generation of commodity-producing SynechocystisPCC6803 strains having high level, controlled expression may be more achievable. en_US
dc.language.iso eng en_US
dc.publisher BioMed Central en_US
dc.relation 309086 en_US
dc.relation.ispartofseries Biotechnology for Biofuels;8:201
dc.subject yellow fluorescent protein (YFP) en_US
dc.subject pCA2.4 en_US
dc.subject gene dosage en_US
dc.subject gene expression en_US
dc.subject synechocystis PCC6803 en_US
dc.title Utilising the native plasmid, pCA2.4, from the cyanobacterium synechocystis sp. strain PCC6803 as a cloning site for enhanced product production en_US
dc.type info:eu-repo/semantics/article en_US
dc.type.supercollection all_ul_research en_US
dc.type.supercollection ul_published_reviewed en_US 2016-02-25T16:49:40Z
dc.description.version PUBLISHED
dc.identifier.doi 10.1186/s13068-015-0385-x
dc.contributor.sponsor ERC en_US
dc.relation.projectid 309086 en_US
dc.rights.accessrights info:eu-repo/semantics/openAccess en_US
dc.internal.rssid 1618934
dc.internal.copyrightchecked Yes
dc.description.status peer-reviewed

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