Nuclear transformation of the versatile microalga Euglena gracilis

Khatiwada, B; Kautto, L; Sunna, A; Sun, A; Nevalainen, H

Nuclear transformation of the versatile microalga Euglena gracilis

Khatiwada, B Kautto, L

Sunna, A Sun, A Nevalainen, H

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Publication Type:: Journal Article
Citation:: Algal Research, 2019, 37 pp. 178 - 185
Issue Date:: 2019-01-01

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Field	Value	Language
dc.contributor.author	Khatiwada, B	en_US
dc.contributor.author	Kautto, L https://orcid.org/0000-0002-4724-8818	en_US
dc.contributor.author	Sunna, A	en_US
dc.contributor.author	Sun, A	en_US
dc.contributor.author	Nevalainen, H	en_US
dc.date.issued	2019-01-01	en_US
dc.identifier.citation	Algal Research, 2019, 37 pp. 178 - 185	en_US
dc.identifier.issn	2211-9264	en_US
dc.identifier.uri	http://hdl.handle.net/10453/136181
dc.description.abstract	© 2018 Elsevier B.V. Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organism requires the development of genetic engineering tools including a method for obtaining genetically stable transformants. In this work, Agrobacterium mediated transformation, biolistic bombardment and electroporation were explored to obtain stable nuclear E. gracilis transformants. Two 3′UTR fragments of the E. gracilis gapC gene were added at the 5′and 3′ ends of the pCambia1302 T-DNA to promote homologous integration of the transforming DNA into the genome. E. gracilis transformants growing on hygromycin plates and expressing the mgfp5 gene coding for green fluorescent protein were obtained from all three approaches. Maintenance of the transforming DNA in the nucleus was confirmed by PCR. Agrobacterium-mediated transformation yielded 10 transformants, biolistic bombardment seven and electroporation one transformant per 10,000 cells plated. Transformants from biolistic bombardment and electroporation were able to transiently express the hptII gene based on their growth on hygromycin containing plates, but this property was lost during repeated rounds of cultivation suggesting lack of (stable) integration of the transforming DNA into the Euglena genome. In contrast, Agrobacterium-mediated transformation produced stable nuclear transformants growing on hygromycin plates even after 12 rounds of cultivation. This work will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.	en_US
dc.relation.ispartof	Algal Research	en_US
dc.relation.isbasedon	10.1016/j.algal.2018.11.022	en_US
dc.title	Nuclear transformation of the versatile microalga Euglena gracilis	en_US
dc.type	Journal Article
utslib.citation.volume	37	en_US
utslib.for	0604 Genetics	en_US
utslib.for	0607 Plant Biology	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
utslib.copyright.status	closed_access
pubs.publication-status	Published	en_US
pubs.volume	37	en_US

Abstract:

© 2018 Elsevier B.V. Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organism requires the development of genetic engineering tools including a method for obtaining genetically stable transformants. In this work, Agrobacterium mediated transformation, biolistic bombardment and electroporation were explored to obtain stable nuclear E. gracilis transformants. Two 3′UTR fragments of the E. gracilis gapC gene were added at the 5′and 3′ ends of the pCambia1302 T-DNA to promote homologous integration of the transforming DNA into the genome. E. gracilis transformants growing on hygromycin plates and expressing the mgfp5 gene coding for green fluorescent protein were obtained from all three approaches. Maintenance of the transforming DNA in the nucleus was confirmed by PCR. Agrobacterium-mediated transformation yielded 10 transformants, biolistic bombardment seven and electroporation one transformant per 10,000 cells plated. Transformants from biolistic bombardment and electroporation were able to transiently express the hptII gene based on their growth on hygromycin containing plates, but this property was lost during repeated rounds of cultivation suggesting lack of (stable) integration of the transforming DNA into the Euglena genome. In contrast, Agrobacterium-mediated transformation produced stable nuclear transformants growing on hygromycin plates even after 12 rounds of cultivation. This work will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.

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http://hdl.handle.net/10453/136181