title: Refinement of the Diatom Episome Maintenance Sequence and Improvement of Conjugation-Based DNA Delivery Methods.
abstract: Conjugation of episomal plasmids from bacteria to diatoms advances diatom genetic manipulation by simplifying transgene delivery and providing a stable and consistent gene expression platform. To reach its full potential, this nascent technology requires new optimized expression vectors and a deeper understanding of episome maintenance. Here, we present the development of an additional diatom vector (pPtPBR1), based on the parent plasmid pBR322, to add a plasmid maintained at medium copy number in Escherichia coli to the diatom genetic toolkit. Using this new vector, we evaluated the contribution of individual yeast DNA elements comprising the 1.4-kb tripartite CEN6-ARSH4-HIS3 sequence that enables episome maintenance in Phaeodactylum tricornutum. While various combinations of these individual elements enable efficient conjugation and high exconjugant yield in P. tricornutum, individual elements alone do not. Conjugation of episomes containing CEN6-ARSH4 and a small sequence from the low GC content 3' end of HIS3 produced the highest number of diatom exconjugant colonies, resulting in a smaller and more efficient vector design. Our findings suggest that the CEN6 and ARSH4 sequences function differently in yeast and diatoms, and that low GC content regions of greater than ~500 bp are a potential indicator of a functional diatom episome maintenance sequence. Additionally, we have developed improvements to the conjugation protocol including a high-throughput option utilizing 12-well plates and plating methods that improve exconjugant yield and reduce time and materials required for the conjugation protocol. The data presented offer additional information regarding the mechanism by which the yeast-derived sequence enables diatom episome maintenance and demonstrate options for flexible vector design.
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, CA, USA; Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Diner
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute , La Jolla, CA , USA.
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Bielinski
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute , La Jolla, CA , USA.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Dupont
authors.affiliation: Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, CA, USA; Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Allen
authors.affiliation: Synthetic Biology and Bioenergy Group, J. Craig Venter Institute , La Jolla, CA , USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Weyman
keywords: DNA delivery; DNA replication; Phaeodactylum; bacteria; conjugation; diatom; episome; genetic tools