Figures and data in Genetic transformation of the dinoflagellate chloroplast

Figures
Tables
Additional files

6 figures, 3 tables and 2 additional files

Figures

Figure 1

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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, chloramphenicol resistance or PsbA) are shown in green. The blue shows the original source of of the genetic material (*E. coli* plasmid and *A. carterae* minicircle). The red arrow showing the position of the mutation in *psbA* that confers resistance to atrazine is marked with ‘M’. Primer sites pAmpPSBA 1: MC-pG-F-II, 2: MC-pG-F, 3: MC-pG-R-II, 4: MC-pG-R; pAmpChl: 5: CAT-R, 6: CAT-F, 7: CAT-FSS. CAT-F-Nest and CAT-R-Nest are immediately adjacent to CAT-F and CAT-R, respectively.

https://doi.org/10.7554/eLife.45292.002

Figure 2

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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 control (PCR with plasmid only), Lane L, Hyperladder 100 bp (Bioline) marker, Lane 1, transformed cell line A5.1 (strong band), Lane 2, transformed cell line A5.2 (faint band), Lane 3, transformed cell line A5.1 but without cells being broken open prior to DNA extraction (no band), Lane 4, wild type (i.e. untransformed cells, no band)). Panel B shows results with pAmpChl. DNA was isolated from cells putatively transformed with pAmpChl, and a PCR reaction performed to amplify a 580 bp region in the plasmid. Lane L, Hyperladder 1 kb (Bioline) marker, Lane P, positive control (PCR with plasmid only), Lane N, negative control (no template), Lane 1, transformed cell line C1A.1, Lane 2, transformed cell line C1A.2, Lane 3, transformed cell line C1A.3. Apparent differences in mobility between bands in lanes P, 1 and 3 are due to gel loading.

https://doi.org/10.7554/eLife.45292.005

Figure 3

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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* only), Lane 2 pAmpChl line (amplifying *psbA* (top band) and CAT (lower band)).

https://doi.org/10.7554/eLife.45292.006

Figure 4

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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, positive control (PCR from plasmid DNA), Lane N, negative control (no template), Lanes 1–3 show products with RNA from three different pAmpPSBA-transformed cell lines (A6.1, A6.2 and A6.3) shown with reverse transcriptase (+RT) and without (-RT). Panel B shows results with pAmpChl. RNA was extracted from cells putatively transformed with pAMPChl, and RT-PCR performed to amplify a 580 bp region. Lane L, Hyperladder 100 bp (Bioline), Lane P, positive control (PCR from plasmid DNA), Lane N, negative control (no template), Lanes 1–3 show products with three different pAmpChl-transformed cell lines (C3.1, C3.2 and C3.3) with reverse transcriptase (+RT) or without (-RT).

https://doi.org/10.7554/eLife.45292.007

Figure 5

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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 secondary Alexa Fluor 405 antibody (blue) showed localization of CAT to the chloroplast (indicated by the overlay image labeled merge).

https://doi.org/10.7554/eLife.45292.008

Figure 6

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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. Lane L, Hyperladder 100 bp (Bioline) marker, Lane N, untransformed cells, Lane 1, transformed cell line A9.1, Lane 2, transformed cell line A9.2, Lane 3, transformed cell line A9.3. Panel A shows PCR to amplify a 560 bp region of pAmpChl. Lane L, Hyperladder 100 bp (Bioline) marker, Lane P, positive control (pAmpChl), Lane N, untransformed cells, Lane 1, transformed cell line C4.1, Lane 2, transformed cell line C4.2, Lane 3, transformed cell line C4.3.

https://doi.org/10.7554/eLife.45292.009

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 (‘untransformed’). Note that cultures from experiments 5–9 were harvested for genetic analysis, and thus the listed survival time is the day of harvesting, labeled with *.

https://doi.org/10.7554/eLife.45292.003

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 lacking the pAmpChl (‘untransformed’). For experiment 1, cells from each plate (three shot with gold particles carrying the plasmid and one with gold particles only) were divided into five separate samples, each incubated at a different chloramphenicol concentration. Note that cultures from experiments C1A, C2, and C3, were harvested for genetic analysis, and thus the listed survival time of lines still alive at that point is the day of harvesting, labeled with *. Experiment C4 was still alive at 57 weeks and is thus marked with +.

https://doi.org/10.7554/eLife.45292.004

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