Abstract
Most genetic transformation protocols for the model diatom Phaeodactylum tricornutum rely on one of two available antibiotics as selection markers: Zeocin (a formulation of phleomycin D1) or nourseothricin. This limits the number of possible consecutive genetic transformations that can be performed. In order to expand the biotechnological possibilities for P. tricornutum, we searched for additional antibiotics and corresponding resistance genes that might be suitable for use with this diatom. Among the three different antibiotics tested in this study, blasticidin-S and tunicamycin turned out to be lethal to wild-type cells at low concentrations, while voriconazole had no detectable effect on P. tricornutum. Testing the respective resistance genes, we found that the blasticidin-S deaminase gene (bsr) effectively conferred resistance against blasticidin-S to P. tricornutum. Furthermore, we could show that expression of bsr did not lead to cross-resistances against Zeocin or nourseothricin, and that genetically transformed cell lines with resistance against Zeocin or nourseothricin were not resistant against blasticidin-S. In a proof of concept, we also successfully generated double resistant (against blasticidin-S and nourseothricin) P. tricornutum cell lines by co-delivering the bsr vector with a vector conferring nourseothricin resistance to wild-type cells.
Highlights
Phaeodactylum tricornutum is one of the most widely used models in diatom research
Two antibiotics are widely used for the selection of transformed diatom cells, both of which were introduced more than two decades ago: phleomycin (e.g., Zeocin/phleomycin D1), together with the respective ShBle resistance gene, as well as nourseothricin and the nat/sat-1 genes (Apt, Grossman & Kroth-Pancic, 1996; Falciatore et al, 1999; Huang & Daboussi, 2017; Sakaguchi, Nakajima & Matsuda, 2011; Zaslavskaia et al, 2000)
The respective resistance proteins were checked for any cross activity with the two other antibiotics in use (Zeocin and nourseothricin), which is required for independent selection with combinations of the antibiotics
Summary
Phaeodactylum tricornutum is one of the most widely used models in diatom research. The genome of P. tricornutum is completely sequenced (Bowler et al, 2008) and different methods for the introduction of transgenes are available, for example, particle bombardment (Apt, Grossman & Kroth-Pancic, 1996), conjugation with bacteria (Karas et al, 2015), or electroporation (Miyahara et al, 2013; Niu et al, 2012; Zhang & Hu, 2014). Genome editing methods like TALEN or CRISPR have been introduced recently, allowing the direct knockout of specific genes (Daboussi et al, 2014; Gruber & Kroth, 2017; Nymark et al, 2016; Serif et al, 2017; Slattery et al, 2018). Despite these important achievements, the availability of antibiotics and the number of selection. Other antibiotics commonly used in protists, like hygromycin and puromycin, were tested in P. tricornutum, but either proved ineffective (hygromycin) or only effective at very high concentrations (e.g. puromycin) (Apt, Grossman & Kroth-Pancic, 1996)
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