Feb 02, 2024
Version 4
Transformation of Diplonema papillatum by electroporation V.4
- Matus Valach1,
- Gertraud Burger1
- 1Université de Montréal, Montreal, Quebec, Canada
Protocol Citation: Matus Valach, Gertraud Burger 2024. Transformation of Diplonema papillatum by electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l28e1xl1y/v4Version created by Matus Valach
Manuscript citation:
Faktorová D, Kaur B, Valach M, Graf L, Benz C, Burger G, Lukeš J. 2020. Targeted integration by homologous recombination enables in situ tagging and replacement of genes in the marine microeukaryote Diplonema papillatum. Environ. Microbiol. 22:3660–3670. (https://doi.org/10.1111/1462-2920.15130)
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 02, 2024
Last Modified: February 02, 2024
Protocol Integer ID: 94591
Keywords: Diplonema, transformation, selection, protist, electroporation, antibiotic resistance, diplonemid
Funders Acknowledgement:
Gertraud Burger
Grant ID: RGPIN-2019-04024
Abstract
Variant protocol for transformation of Diplonema papillatum by electroporation using a "home-made" transformation buffer. The procedure was devised based on previously published protocols by Kaur et al. (DOI: 10.1111/1462-2920.14041) and Dyer et al. (DOI: 10.3791/54342). For additional details, see also Faktorová et al. (DOI: 10.1111/1462-2920.15130).
Materials
MATERIALS
Sodium Phosphate monobasic
Glucose
KCl
CaCl2
MgCl2
BSA
Sucrose
HEPES
EDTA
Inosine triphosphate
G418 (geneticin sulfate)BioshopCatalog #GEN418
Before start
Perform a simple test of antibiotic resistance of wild-type cells in the chosen culture conditions, e.g., temperature (16 vs 20 vs 27 °C), medium composition (e.g., horse serum vs fetal bovine serum) or antibiotic supplier. Into a 24-well plate, distribute 1.5 mL medium per well and add the antibiotic at several different concentrations (e.g., for G418, choose 0, 50, 75, 100, 150, and 200 µg/mL). This arrangement (6 columns, each with a different antibiotic concentration) allows to perform three WT replicates together with one positive control resistant to the antibiotic of choice. Inoculate 1–5×105 cells per well and let the cells grow for 3–4 days, then examine the extent of growth. The lowest antibiotic concentration at which the WT cells do not grow is then used for the selection.
For example, when cultivating Diplonema papillatum in a horse serum-based medium and using G418 (Bioshop; potency min. 650 µg/mg), 100 µg/mL is the threshold value at 20 °C, but >125 µg/mL is needed for efficient selection at 16 °C.
Prepare the transformation (cytomix-like) buffer.
| A | B | |
| Component | Final concentration | |
| HEPES pH7.5 | 25 mM | |
| KCl | 25 mM | |
| CaCl2 | 0.15 | |
| NaH2PO4 pH7.5 | 10 | |
| MgCl2 | 2.5 | |
| EDTA | 1 | |
| glucose | 30 mM (0.5%) | |
| sucrose | 145 mM (4.35%) | |
| bovine serum albumin (BSA) | 0.1 mg/mL | |
| inosine triphosphate (ITP) [or hypoxanthine] | 1 mM |
Note
The addition of ITP (or hypoxanthine) is optional (alternatively, ATP can be used). If preparing a large volume of the buffer, make aliquots and store them at −70 °C until further use.
Inoculate Diplonema cells at 1–2×105 /mL into 100 mL OSS medium supplemented with 0.05% tryptone and let them grow for 2–3 days.
Note
Cell density is ~5-times higher when cultivating Diplonema cells in a medium containing 0.05% tryptone compared to a medium without such supplementation. This improves survival after the pulse and is especially useful for high-voltage conditions (see below), which seems to favorise homologous integration. The resulting amount of cells is usually sufficient for 4–6 transformations. Therefore, if performing additional transformations, scale-up the cultivation volume. (If performing selection at 20 °C, pre-culture is done at this same temperature.)
Harvest the cells while they are in the late exponential phase (optimal density 8×106–2×107 /mL) by centrifugation (2,000×g, 5 min, 4 °C).
Note
The number of cells required depends on the pulse parameters (see the step #7 below). For high voltage conditions, 4×108 cells means that more cells will survive the pulse and the probability of a successful transformation increases. For low voltage conditions, much lower number of cells per transformation should be used because higher cell densities generally result in a quasi-totality of transformants having the transformed DNA construct inserted at non-homologous locations. Conversely, if homologous integration is of little interest, subjecting a high number of cells to the pulse is beneficial since many more clonal cell lines can be obtained.
Resuspend the pelleted cells in OS (i.e., medium without the serum), then transfer them into 1.5-mL tubes and centrifuge (2,000×g, 5 min, 4 °C).
Note
If harvesting cells from 100 mL of OSS, resuspend the cell pellet in 2–3 mL OS and transfer into two 1.5-mL tubes.
Repeat the washing with OS once more, then aliquot the cells into tubes, so that after the final centrifugation, each pellet contains 1–4×108 cells. Remove as much OS buffer as possible. From this point on, keep the cells on ice.
Resuspend the pellet in ice-cold 200 µL transformation buffer (see the recipe above), immediately centrifuge (4 °C, 1,000×g, 1 min), and discard the supernatant.
Note
If not proceeding to electroporation immediately, keep the cells on ice without any buffer. Note that any delay decreases transformation efficiency, but a pause of 5–10 min can be accomodated if necessary.
Resuspend the pellet in ice-cold 100 µL transformation buffer supplemented with 1–4 µg linearized DNA (e.g., a PCR product or a restriction fragment of a plasmid).
Note
Optimally, add the DNA in a volume of 5 µL or less. To the negative control, add the same volume of the buffer used to solubilize the linearized DNA (e.g., 10 mM Tris pH8.0).
Immediately transfer the cell suspension into an electroporation cuvette (0.2 mm), which has been pre-cooled on ice.
Wipe the cuvette to remove moisture, quickly transfer the cuvette into an electroporation apparatus (e.g., Gene Pulser Xcell from Bio-Rad), and apply the pulse.
Pulse parameters:
- 1,500 V, 0.4 ms (also referred to here at "high voltage"); or
- 140 V, 1,400 µF ("low voltage").
Note
Cell line selection is more straightforward and clear-cut for the option 1 (high voltage) and we observed that a higher proportion of transformants has had the construct integrated at the intended locus (~60%), but the number of independent cell lines is limited (up to 5 independent cell lines have been obtained, but usually only about 2).
In contrast, cell survival is much more substantial in the option 2 (low voltage) and may be preferred when numerous clones are required (up to 45 independent cell lines have been obtained). However, as indicated above, transformants tend to integrate the construct at a non-homologous location much more prominently.
Immediately after the pulse, put the cuvette back on ice, add 1 mL cold (1–5 °C) OSS, and resuspend the cells.
Transfer the cell suspension into a well of a 24-well (or 48-well) plate. Distribute the pulsed cell suspension into 24–48 wells (depending on the expected or desired number of independent clones, but the higher the number of wells, the more likely it is that a pure clonal cell line will quickly be obtained). Add additional OSS into each well (~1 and ~0.5 mL when using 24- and 48-well plates, respectively). Cultivate for 5–8 h without selection.
Prepare OSS with the antibiotic of choice at a concentration that is double of the selection concentration (e.g., 200 µg/mL G418 if the final selection concentration is to be 100 µg/mL). To each well with pulsed cell suspension in OSS, add an equal volume of this medium. The final volume is usually 1.6–2 mL (24-well plates) or 0.8–1 mL (48-well plates).
Note
Make sure that the final concentration of the antibiotic is as determined by the resistance test. Optionally, keep a single well without the antibiotic (i.e., add an equal volume of just OSS) to keep track of the recovery of the pulsed cells. This is especially useful when applying a high voltage pulse to less than 5×107 cells.
Let the cells grow for 2–4 days. Observe the cells in the plates under a microscope to check their growth. If there is visible growth, i.e., cells actively swimming (as opposed to floating passively) in the 'column', transfer an aliquot of these swimming cells into a new plate with a 1.5–2× higher concentration of the antibiotic (e.g., if using G418 at 100 µg/mL, this well population passaging should be done at 150–200 µg/mL). After a growth for additional 5–9 days, start analyzing well populations or make conserves for later analyses.
Note
Passaging the cells through a medium with a higher concentration of the antibiotic ensures that only truly resistant clones (i.e., those expressing the antibiotic resistance-conferring gene at a sufficient level) are selected.
If a well population is a mixture of cells containing a wild-type allele and a correctly integrated DNA construct, perform 10× serial dilutions of cells from each selection well into a new plate with fresh medium (if using G418, usually at 100–150 µg/mL) to ensure that truly independent cell lines are selected. This phase may take up to 3 weeks in total.