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.
Highlights
Diatoms play a critical role in marine ecosystems and global carbon cycling
We aimed to determine which combinations of sub-sequences within the yeast CEN6-ARSH4-HIS3 were sufficient to allow episome maintenance
While dissecting the yeast-derived sequence required for episome maintenance in P. tricornutum, we found that pPtPBR derivatives containing the CEN6, ARSH4, and a truncated version of the HIS3 sequence encompassing the low GC portion in a contiguous sequence resulted in a higher number of exconjugant colonies than versions containing the entire HIS3 sequence (Figure 2)
Summary
Diatoms play a critical role in marine ecosystems and global carbon cycling. They are excellent candidates for bioproduction of valuable commercial compounds and renewable energy resources, as they display rapid growth rates across a range of environmental conditions. The diatom Phaeodactylum tricornutum has emerged as an important model for examining diatom biological processes and commercial potential and as a test strain to develop new genetic tools that can be expanded into other diatom species (Apt et al, 1996; Lopez et al, 2005; Siaut et al, 2007; Bozarth et al, 2009). P. tricornutum was shown to stably maintain engineered diatom episomes delivered to the diatoms via bacterial conjugation, which represents a major advancement in diatom tool development but requires further optimization (Karas et al, 2015)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
