1. 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

2. 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

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

4. 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.

5. 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

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

7. 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.

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

9. 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

10. 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.

11. 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.

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

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

14. 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

15. 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

16. 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

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

18. 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

19. Bogumil Karas Andrew Allen Phil Weyman Chris Dupont aallen@jcvi.org
Andrew Allen
Chris Dupont
Phil Weyman

20. 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-
Funding:

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