Genetic transformation of the dinoflagellate chloroplast
- University of Cambridge, United Kingdom
- King Abdullah University of Science and Technology (KAUST), Saudi Arabia
- Sunway University, Malaysia
- University College London, United Kingdom
Figures
Figure 1
Artificial minicircle design.
Left, pAmpPSBA. Right, pAmpChl. Origins of replication (ori for E. coli plasmid, core region for A. carterae minicircle) are shown in red. Protein-coding genes (encoding ampicillin resistance, …
Figure 2
Presence of vectors in transformed A. carterae.
Panel A shows results with pAmpPSBA. DNA was isolated from cells putatively transformed with pAmpPSBA, and a PCR reaction performed to amplify a ~ 200 bp region in the plasmid. Lane P, positive …
Figure 3
Relative copy number of psbA and pAmpChl minicircles.
DNA was isolated from cells transformed with pAmpChl, and a PCR reaction performed to amplify either psbA or CAT. Lane L, Hyperladder 1 kb (Bioline) marker, Lane 1, wildtype cells (amplifying psbA …
Figure 4
Transcription of minicircles.
Panel A shows results with pAmpPSBA. RNA was extracted from cells putatively transformed with pAmpPSBA, and RT-PCR performed to amplify a ~ 500 bp region. Lane L, Hyperladder 1 kb (Promega), Lane P, …
Figure 5
A chloroplast localization for chloramphenicol acetyl transferase.
Immunofluorescence microscopy using the A. carterae pAmpChl line. Cells (brightfield) showed significant autofluorescence in the chloroplast (red). A primary antibody specific for CAT with a …
Figure 6
Long-term stability of transformation.
DNA was isolated from cells putatively transformed with pAmpPSBA or pAmpChl and maintained under selection 3 months (Experiments A9 and C4). Panel A shows PCR to amplify a 200 bp region of pAmpPSBA. …
Tables
Table 1
Biolistic transformation of A. carterae with pAmpPSBA.
Each experiment was carried out in triplicate, thus producing three potentially transformed lines. In addition, one line of cells was subjected to biolistic bombardment, but without the pAmpPSBA …
| Experiment | Rupture disk (p.s.i.) | Atrazine concentration (µg ml−1) | Survival untransformed (days) | Mean survival pAmpPSBA (days) |
|---|---|---|---|---|
| A1 | 1100 | 2.5 | 13 | 13 |
| A2 | 1350 | 2.5 | 13 | 16 |
| A3 | 1350 | 2.5 | 13 | 15 |
| A4 | 1550 | 2.5 | 13 | 17 |
| A5 | 1550 | 2.5 | 12 | 20* |
| A6 | 1550 | 2 | 7* | 7* |
| A7 | 1550 | 2 | 13 | 13* |
| A8 | 1550 | 2 | 12 | 15* |
| A9 | 1550 | 1 | 3 months* | 3 months* |
Table 2
Biolistic transformation of A. carterae with pAmpChl.
Each experiment was carried out in triplicate, thus producing three potentially transformed lines. In addition, one line of cells was subjected to biolistic bombardment, but with gold particles …
| Experiment | Rupture disk (p.s.i.) | Chloramphenicol concentration (µg ml−1) | Survival untransformed (days) | Mean survival pAmpChl (days) |
|---|---|---|---|---|
| C1A | 1550 | 10 | 15 | 35* |
| C1B | 1550 | 20 | 15 | 17 |
| C1C | 1550 | 30 | 15 | 15 |
| C1D | 1550 | 40 | 15 | 15 |
| C1E | 1550 | 50 | 13 | 15 |
| C2 | 1550 | 10 | 13 | 13* |
| C3 | 1550 | 10 | 14* | 14* |
| C4 | 1550 | 20 | 16 | 57 weeks + |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Amphidinium carterae) | A. carterae CCMP1314 | Culture Collection of Marine Phytoplankton | CCMP1314 | |
| Genetic reagent (vector) | pAmpChl | this paper, synthesized by GeneArt | Full sequence provided in supplemental data. | |
| Genetic reagent (vector) | pAmpPSBA | this paper | Full sequence provided in supplemental data. | |
| Antibody | Rabbit anti-Chloramphenicol-acetyl-transferase, polyclonal | Antibodies-Online | Antibodies-Online Cat# ABIN285051, RRID:AB_10781219 | 1:500 in f/2 medium with 5% BSA, 1 hr. |
| Antibody | Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 405, polyclonal | ThermoFisher | Thermo Fisher Scientific Cat# A-31556, RRID:AB_221605 | 1:1000 in f/2 medium with 5% BSA, 1 hr. |
| Commercial assay or kit | DNAdelTM Gold Carrier Particles Optimized for Plasmid Delivery | Seashell Technology | Seashell Technology Cat# S550d |
Additional files
-
Supplementary file 1
Primer sequences and vector sequences.
- https://doi.org/10.7554/eLife.45292.010
-
Transparent reporting form
- https://doi.org/10.7554/eLife.45292.011
