title: Designer diatom episomes delivered by bacterial conjugation.
abstract: Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Bogumil J
authors.initials: BJ
authors.lastname: Karas
authors.affiliation: 1] Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA [2] Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92037, USA.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Diner
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Stephane C
authors.initials: SC
authors.lastname: Lefebvre
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: McQuaid
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Alex P R
authors.initials: AP
authors.lastname: Phillips
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Chari M
authors.initials: CM
authors.lastname: Noddings
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Brunson
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Ruben E
authors.initials: RE
authors.lastname: Valas
authors.affiliation: National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Jelena
authors.initials: J
authors.lastname: Jablanovic
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Jeroen T F
authors.initials: JT
authors.lastname: Gillard
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Beeri
authors.affiliation: National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: John I
authors.initials: JI
authors.lastname: Glass
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Hutchison
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Hamilton O
authors.initials: HO
authors.lastname: Smith
authors.affiliation: 1] Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA [2] Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: J Craig
authors.initials: JC
authors.lastname: Venter
authors.affiliation: 1] Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA [2] Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92037, USA.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Allen
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Dupont
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Weyman
mesh_terms: D003227:Conjugation, Genetic; D004247:DNA / Q000235:genetics; D017377:Diatoms / Q000235:genetics*; D018274:Electroporation; D004926:Escherichia coli / Q000502:physiology*; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D011092:Polyethylene Glycols; D004247:DNA