Refinement of the diatom episome maintenance sequence and improvement of conjugation-based DNA delivery methods

TitleRefinement of the diatom episome maintenance sequence and improvement of conjugation-based DNA delivery methods
Publication TypeJournal Article
Year of Publication2016
AuthorsDiner RE, Bielinski VA, Dupont CL, Allen AE, Weyman PD
JournalFrontiers in Bioengineering and Biotechnology
Date Published2016/08
Type of ArticleMethods
ISBN Number2296-4185
Keywordsdiatom,Bacteria,conjugation,Genetic tools,Episome,DNA delivery,DNA Replication,Phaeodactylum

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 E. 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 P. tricornutum. While various combinations of these individual elements enable efficient conjugation and high ex-conjugant 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 ex-conjugant 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 higher-throughput option utilizing 12-well plates, and plating methods that improve ex-conjugant 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.

Short TitleRefinement of Diatom Episome Maintenance and Delivery
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