Designer diatom episomes delivered by bacterial conjugation

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.

Diatoms form associations with E. coli. Scanning electron microscopic (SEM) images of T. pseudonana (A and B) and P. tricornutum (C and D) cells after the 90 minute conjugation incubation at 30° C with E. coli containing p0521s and pTA-MOB. Image C shows a dividing P. tricornutum cell that is in the process of changing to the ovoid morphotype. Scale bar indicates 1 µm.
Results of sequencing p0521s plasmids rescued from P. tricornutum exconjugants. Various deletions (e.g. C8 and C9, red star with blue highlighted regions) and insertions (e.g. c18 and c19, green triangle) were detected by Sanger DNA sequencing.

Supplementary Figure 5
Outline of the experimental setup to test p0521s and p0521 maintenance in P. tricornutum lines grown in the absence of antibiotic selection. P. tricornutum lines containing p0521s were grown with or without selection for 28 days during which time they were serially diluted one hundredfold at three different times (A). After 28 days, culture was plated to obtain single colonies on non-selective medium (B) and patched on non-selective medium (C). Colonies were replica patched on selective (phleomycin 20 µg ml -1 ) solid L1 medium (D). E. Stability of plasmid p0521 in P. tricornutum lines after growth in seawater medium with or without antibiotic selection. The experiment was performed as outlined in part A, but for a total of 18, 28, or 38 days. F. Episome rescue from single P. tricornutum p0521 lines sub-cultured without (lanes 1-3) or with (lanes 4-6) antibiotic selection was performed after 18 (F) or 38 (G) days. After subculture, P. tricornutum lines were selected on solid medium with antibiotic selection and episomes were rescued and separated by agarose gel electrophoresis.
Plasmid p0521 replicates as a circle in P. tricornutum and with copy number equivalent to native chromosomes. A. DNA isolated from P. tricornutum lines containing p0521 was untreated or treated with Plasmid-safe exonuclease (Epicentre) and separated by agarose gel electrophoresis. Arrow indicates P. tricornutum genomic DNA that was degraded by the exonuclease treatment (+) but still present in control treatment (-). B. After transformation of the treated or control DNA from part A into E. coli, equivalent numbers of colonies were observed. C. Results of qPCR with DNA isolated from P. tricornutum lines containing p0521. See Fig. 2E for legend. P. tricornutum genomic DNA was extracted from wild type (WT) or p0521s ex-conjugant lines (c3,c9,c14,and c18,see Extended Data Fig. 3A) and digested with ClaI that cuts a single time within p0521s. To avoid ClaI Dam methylation in E. coli-isolated plasmid controls, p0521s isolated from E. coli was digested with RsrII that cuts a single time within the plasmid. A. DNA (30 µg) was separated by agarose gel electrophoresis for 18 hr at 25 V to resolve higher molecular weight DNA and stained with ethidium bromide. The plasmid band is visible in the ethidium bromide-stained gel (arrow). B. Gels were blotted and hybridized with a probe to the ShBle gene. Only one band was observed on the ShBle-probed blot corresponding to the linearized plasmid position and consistent with lack of insertion of the plasmid elsewhere in the chromosome.
Plasmid pTpPuc3 rescued from T. pseudonana exconjugant lines. DNA from 16 T. pseudonana exconjugant lines was extracted and transformed to E. coli for subsequent isolation and separation by agarose gel electrophoresis. Lanes marked with "M" indicate 1-kb ladder (NEB) and with "C" indicate pTpPuc3 plasmid control.