Development of Replicating Plasmids Delivered to Diatoms by Bacterial Conjugation Rachel E. Diner UC San Diego, Scripps Institution of Oceanography The J. Craig Venter Institute Andrew Allen Laboratory

Diatom Molecular Tools Genetic transformation and manipulation Model diatom species: P. tricornutum and T. pseudonana Examples: Fluorescent protein localization, Gene silencing/knockouts Allen et al. 2011 Limitations: Inefficient DNA delivery methods (e.g. biolistic particle delivery): labor and cost intensive Inconsistencies due to random and/or fragmented genomic integration No autonomous replicating plasmid more consistent expression, better control of expression vectors ability to remove from line

Study Aims: Develop an improved DNA delivery method Create the first episomal vector, including identification of plasmid maintenance factors Apply to multiple diatom species

Development of an improved DNA delivery method Bacteria (e.g. E. coli) Conjugative Plasmid T4SS OriT Bacteria Plasmid Yeast nucleus Mammalian Cell Traditional bacterial conjugation requires an F-pilus, the machinery for which is encoded on the conjugative plasmid. If the conjugative plasmid contains an origin of transfer it will be transferred to the recipient. This is a common process in bacteria, but has also been shown to occur between E. coli and eukaryotic cells, including yeast and mammalian cells. We have shown that this can also occur between E. Coli and Pt.

Conjugation-based DNA delivery to P Conjugation-based DNA delivery to P. tricornutum using a two-plasmid system Allows Cargo Plasmid to transfer E. coli Cargo Plasmid 6-16 kb Conjugative Plasmid 60 kb OriT Cargo To transform Pt, we used a two plasmid system including both a conjugative and cargo plasmid. If both plasmids contain an origin of transfer, they will both be transferred to the recipient. We used the conjugative plasmid pRL443, which contains an origin of transfer and all conjugative machinery, but does not contain elements required for replication in Pt, so it would not be maintained as an autonomous plasmid. If the E.coli contains another plasmid with an origin of transfer, that plasmid will also be transferred. In the case of p0521s, which contains the yeast elements enabling replication, the plasmid can then be maintained in Pt. The plasmid can then be used to express genes of interest (which we have demonstrated) ShBle (Pt selectable Marker) P. tricornutum Karas et al. (2015) Designer diatom episomes delivered by bacterial conjugation. Nature Communications

High transformation efficiency via bacterial conjugation Optimal Method (see Karas et al. 2015 for specific protocols): 1hr 30min at 30°C Specific concentration of E. coli and Pt cells Re-plate on selection after 2 days

High plasmid delivery efficiency via bacterial conjugation P. tricornutum and E. coli Cargo Plasmid + +(No OriT) + + - - Conjugative Plasmid Plasmid extraction from diatom recovery E. coli transformation verification Plasmid-safe Gel imaging Stability/Copy # Karas et al. (2015), Nat. Comm.

Transformation of T. pseudonana T. pseudodonana and E. coli Cargo plasmid + Conj. Plasmid Karas et al. (2015), Nat. Comm.

How is the delivered plasmid maintained? Initial hypothesis: Pt “centromere” could drive plasmid replication Tested various regions of Pt genomic DNA Plasmid p0521s (~16kb, assembled in yeast) replicated in Pt and recovered in E. coli Long-term stability: maintained stably for >1 year Pt DNA Yeast elements E. coli elements

Pt DNA improves efficiency, but is not required for plasmid maintenance Pt DNA Regions 1 & 2 Region 1 Only Region 2 Only Pt Regions 1 & 2 removed Karas et al. (2015), Nat. Comm.

Yeast elements (not Pt elements) enable plasmid replication PUC19 backbone + + - - + - Pt DNA Elements Yeast Replication Elements Same results in T. pseudonana Karas et al. (2015), Nat. Comm.

New Vectors for Diatom Genetic Manipulation S. elongatus DNA (~65KB) Stable maintenance of large pieces of Foreign DNA Expression of fluorescently labelled proteins

New Vectors for Diatom Genetic Manipulation Small, high copy number Vector flexibility

Yeast Replication Elements Cen-Ars-His Why do the yeast elements allow diatom plasmid replication? Yeast Replication Elements Cen-Ars-His Cen Centromere ~100bp ~10% GC Content Ars Origin of Replication ~377bp ~28% GC Content His Histidine Marker ~800bp ~40% GC Content Low-GC content region

Individual yeast elements are not sufficient for replication Plasmid Maintained Cen + Ars + His No CAH Cen Only Ars Only His Only Cen + Ars Cen & Ars spatially separated

Yeast Replication Elements Cen-Ars-His ~200bp ~28% GC Content Cen Centromere ~100bp ~10% GC Content Ars Origin of Replication ~377bp ~28% GC Content His Histidine Marker ~800bp ~40% GC Content centromere- region of DNA segregation ARS- initiation of DNA replication Low-GC content region

Low-GC content defines the “functional region” of His Cen + Ars + His Ars + His Cen + Ars + 100bp His Cen + Ars + 200bp His

Summary Efficient, low-cost transformation method Episome replication enabled by yeast elements Multiple species: P. tricornutum and T. pseudonana Current/Future Projects: Sequence determinants of Pt plasmid and chromosomal replication Horizontal gene transfer Artificial chromosome development

Bogumil Karas Andrew Allen Phil Weyman Chris Dupont aallen@jcvi.org Bogumil@Designermicrobesinc.com Andrew Allen aallen@jcvi.org Chris Dupont cdupont@jcvi.org Phil Weyman pweyman@jcvi.org

Funding: Karen Beeri (JCVI) Chari Noddings (JCVI/UT) Synthetic Biology and Bioenergy Group Environmental and Microbial Genomics Group Bogumil Karas (Designer Microbes Inc.) Phil Weyman (JCVI) Chris Dupont (JCVI) Andrew Allen (JCVI/SIO) Stephane Lefebvre (JCVI) Jeff McQuaid (JCVI/SIO) Jelena Jablanovic (JCVI) Karen Beeri (JCVI) Chari Noddings (JCVI/UT) Patrick Brunson (JCVI/SIO) Rachel Diner- rdiner@ucsd.edu Funding:

Yeast DNA Elements allow plasmids to replicate in diatoms cargo Yeast Elements: Centromere Origin of Replication Histidine (marker)