Construction of the BiG Mito-Split-GFP strain

Summary

The genomes of S. cerevisiae, both nuclear and mitochondrial, undergo homologous recombination. This feature allows to easily remove sequences of or introduce mutations into a specific gene. The mitochondrial genome encodes only eight peptides which are either components of the respiratory chain complexes (7 of them) or of the mitochondrial ribosome. The ability of yeast cells to survive without mtDNA is another feature that allows for its modification. Recombination of a DNA fragment into the mitochondrial genome requires, however, that it resides inside the mitochondrial network. Thus, the plasmid DNA that we call: the "donor" and which is encoding the fragment that we wish to insert into the mitochondrial genome that we call: the "recipient", must be transferred inside the mitochondria. We use for this purpose, the biolistic transformation, an experimental technic which is extensively described in detail in [1] and, therefore not described herein. Below, we present a protocol for the construction of the BiG Mito-Split-GFP strain by cytoduction of the mitochondrial genomes between strains with different nuclear genetic backgrounds, which can be used for other mtDNA modifications: gene deletion or site-directed mutagenesis. The construction of this strain required many steps, because the GFP_β1-10 gene sequence had to be integrated in such a way as to not disturb respiratory functions. First, (Step 1 and 2) we obtained a strain in which all mitochondrial genes were present and functional and, at the same time, one mitochondrial locus was made available to integrate the GFP_β1-10 gene (Step 3) by homologous recombination with the donor. Note that the mitochondrial locus that was made available for integration was still flanked by its endogenous 5’- and 3’ regulatory sequences.

Before you begin:

1. Prepare solid and liquid media as described in Materials.

2. Plate the strains from the stocks onto YPGA solid medium and grow them according to standard procedures [2]. After one day of incubation on YPGA medium, subclone the atp6::ARG8m strain on W0 lacking arginine (W0-arg) plate in order to select cells maintaining the mitochondrial genome (r⁺). The genotype of strains used in each step is listed in Table 1. The scheme of the procedure is included in the supplementary Fig. S2 of the eLIFE article describing the BiG Mito-Split-GFP strain engineering [3].

Step 1 - Construction of a strain with a mitochondrial genome devoid of the ATP6 gene and with deletion of a fragment of the COX2 gene (RKY83). This strain was necessary to select the integration of the ATP6 gene into the COX2 locus in the following step 2.

1. Grow a starter cultures of MR10 and YTMT2 strains in 5 mL of W0-arg and YPGA media, respectively, to a density of OD₆₀₀ = 1-2. In the Eppendorf tube, mix both strains (in order to cross them) in a cell ratio of 3:1 in 50 µL of YPGA, then put the drop of cells mixture onto a plate with W0-arg 5 % glucose. Incubate in an incubator at 28 °C for two days. In this cross, heterocaryons are formed due to the kar1-1 mutation in the YTMT2 strain (see Table 1) which blocks karyogamy but does not block cytogamy, allowing the mitochondrial networks of both strains to fuse,mix and recombine their mitochondrial genomes. In the first division, one nucleus is inherited to daughter cells, while obtaining fully homoplasmic colonies, in terms of mtDNA, requires a minimum of 20 divisions. In such a crossing experiment only 5 % of the colonies are diploids.

3. Transfer the cells from the cross into 50 mL of YPGA 5 % glucose medium and grow for one day at 28 °C.

4. Next day, transfer 100 µL of this culture to 50 mL of fresh YPGA 5 % glucose medium and grow for another day at 28 °C.

5. Next day, measure OD₆₀₀ and plate onto 10 plates of W0-arg 5 % glucose (~100 colonies per plate) and incubate at 28 °C for two days. Use the following formula to convert the optical density to the number of cells:

Number of cells = OD₆₀₀ x 1.2 x 10⁷

6. Mark the plate orientation on each of the 10 plates containing the cytoductants. To identify the colonies bearing the recombinant r⁺ cox2-62 atp6::ARG8m mtDNA, replica plate cytoductants to plates: W0-arg, W0-trp (lacking tryptophane), YPGA with YTMT2 strain and YPGA with SDC30 strain plated at 10⁸ density to cross cytoductants with these two tester strains. Make sure that the outline of the colonies is visible on each plate after the replica-plating. 

7. After one day of incubation of the crossing plates at 28 °C replica plate them on the YPGlyA plates. Incubate plates two days at 28 °C. 

8. Analyze the phenotype of each individual cytoductant on each medium. The correct clones are unable to grow on W0-trp plates (MR10 strain nucleus selection) but are able to grow on W0-arg and to produce respiring clones after crossing with the SDC30 but not with the YTMT2 strain.

Step 2 - Construction of the atp6::ARG8m strain in which the ATP6 gene sequence was integrated in between the regulatory sequences of the COX2 gene at COX2 locus (RKY112).

1. Design the DNA fragment encoding the ATP6 gene flanked by 5'- and 3' UTRs of COX2 gene ending with the EcoRI restriction enzymes sites at both ends (Fig. 1). Order the synthesis of this DNA fragment.

2. Cut off the _COX2ATP6_COX2 DNA fragment from received plasmid with EcoRI enzyme and clone it into the pJM2 plasmid encoding the wild-type COX2 gene and its regulatory sequences [4]. By doing so, the pRK49 plasmid was obtained.

3. Introduce the pRK49 into the r^o DFS160 Mat α strain by the method of biolistic transformation according to the protocol described in [1]. Incubate plates at 28 °C for 5-6 days. 

4. Mark the orientation of the plates with transformants and replica plate them onto YPGA and onto YPGA plates with NB40-3C strain plated at 10⁸ density for crossing. Incubate plates for one day at 28 °C.

5. Replicate the crossing plates on the YPGlyA medium plates – make sure to keep visible the outline of the colonies after the replica plating. Incubate the plates at 28 °C for 2 days. If a plasmid encoding the _COX2ATP6_COX2–COX2 gene has been introduced into the mitochondria as a result of biolistic transformation, respiring colonies will appear on the YPGlyA plate.

6. Return back to the YPGA plates containing the transformants and subclone cells from the region that contain the mitochondrial transformant to YPGA plates in such a way as to have single colonies. Incubate plates at 28 °C for 2 days.

7. Repeat steps 4, 5 and 6 until obtaining single colonies complemented by NB40-3C strain - purified mitochondrial transformants, the so-called r ^-S (the RKY89, in the procedure for obtaining the RKY112 strain, Table 1). For the following crossing, work at least with two independent purified mitochondrial transformants. 

9. Cross the r ^-S strain with the r⁺ cox2-62 atp6::ARG8m (RKY83) strain and let the genomes to segregate exactly as described in Step 1, points 1 to 5. To identify clones bearing the recombinant mtDNA, replicate the colonies (minimum 1000) onto plates: W0-arg, W0-trp, YPGlyA and YPGA with RKY25 strain (mit^- atp6-L247R) plated at 10⁸ density for crossing. If the ATP6 gene is functional from the regulatory sequences of COX2 gene, the correct recombinant clones will grow on YPGlyA and W0-arg (which indeed was the case – the RKY112) but not be able to grow on W0-trp. Crossing with RKY25 strain was done to confirm the integration of ATP6 gene in the COX2 locus in case when such ectopic expression would not be functional. The integration of ATP6 sequence would complement the respiratory deficiency of atp6-L247R mutant. 

Step 3 - Construction of the RKY176 strain.

1. Design the DNA fragment containing the GFP_β1-10 gene flanked by 5 'and 3' UTRs of ATP6 gene and ended by the BamHI-EcoRI restriction enzymes sites (see the sequence in supplementary data of the eLife original article [3]). Order the synthesis of this DNA fragment.

2. Cut off the _ATP6GFP_β1-10ATP6 DNA fragment from the ordered plasmid with BamHI-EcoRI enzymes and clone it into the pJM2 plasmid encoding the wild-type COX2 gene flanked by its regulatory 5’- and 3’ sequences [4], thereby obtaining the pRK67 plasmid.

3. Introduce the pRK67 into the r^o DFS160 Mat α strain by the method of biolistic transformation [1]. Incubate plates at 28 °C for 5-6 days. The mitochondrial r ^-S transformants were purified as described in Step 2: points 4 to 7 (the RKY172 strain in the procedure for obtaining the BiG Mito-Split-GFP strain, Table 1).

4. Cross the r ^-S strain RKY172 with the atp6::ARG8m _COX2ATP6_COX2 (RKY112) strain as described in the Step 1 of this protocol, however, after the second passage of cytoductants, spread them on YPGA plates. To identify clones bearing the recombinant mtDNA, replicate the colonies (minimum 1000) into plates: W0-arg, W0-trp and YPGlyA. Colonies unable to grow on W0-arg medium have integrated the GFP_β1-10gene at the ATP6 locus of mtDNA replacing the ARG8m gene sequence.

Step 4 – Restoring the nuclear ADE2 locus into RKY176 (BiG Mito-Split-GFP) strain. This was necessary in order to eliminate interfering fluorescence emission of the vacuole due to accumulation of a pink adenine precursor and is described in the original eLife article [3].

Materials 

Growth Media 

YPGA (rich glucose): 1 % (w/v) Bacto yeast extract, 1 % (w/v) Bacto Peptone, 2 % (w/v) or 5% (w/v) glucose, 40 mg/L adenine; YPGlyA: 1 % (w/v) Bacto yeast extract, 1 % (w/v) Bacto Peptone, 2 % (v/v) glycerol, 40 mg/L adenine, CSM (complete synthetic medium): 2 % (w/v) or 5 % (w/v) glucose, yeast nitrogen base 1,7 g/L, ammonium sulfate 5 g/L, 40 mg/L adenine, and selective drop-out amino acid mix. The liquid media were solidified by addition of 2 % (w/v) of Bacto Agar. 

Genotypes of yeast strains.

Sequence of the DNA fragment of ATP6 flanked by the 5’- and 3’ regulatory sequences of COX2 gene:

C G G A A T T C T A A A T T T T A A T T A A A A G T A G T A T T A A C A T A T T A T A A A T A G A C A A A A G A G T C T A A A G G T T A A G A T T T A T T A A A A T G T T T A A T T T A T T A A A T A C A T A T A T T A C A T C A C C A T T A G A T C A A T T T G A G A T T A G A C T A T T A T T T G G T T T A C A A T C A T C A T T T A T T G A T T T A A G T T G T T T A A A T T T A A C A A C A T T T T C A T T A T A T A C T A T T A T T G T A T T A T T A G T T A T T A C A A G T T T A T A T C T A T T A A C T A A T A A T A A T A A T A A A A T T A T T G G T T C A A G A T G A T T A A T T T C A C A A G A A G C T A T T T A T G A T A C T A T T A T A A A T A T G C T T A A A G G A C A A A T T G G A G G T A A A A A T T G A G G T T T A T A T T T C C C T A T G A T C T T T A C A T T A T T T A T G T T T A T T T T T A T T G C T A A T T T A A T T A G T A T G A T T C C A T A C T C A T T T G C A T T A T C A G C T C A T T T A G T A T T T A T T A T C T C T T T A A G T A T T G T T A T T T G A T T A G G T A A T A C T A T T T T A G G T T T A T A T A A A C A T G G T T G A G T A T T C T T C T C A T T A T T C G T A C C T G C T G G T A C A C C A T T A C C A T T A G T A C C T T T A T T A G T T A T T A T T G A A A C T T T A T C T T A T T T C G C T A G A G C T A T T T C A T T A G G T T T A A G A T T A G G T T C T A A T A T C T T A G C T G G T C A T T T A T T A A T G G T T A T T T T A G C T G G T T T A C T A T T T A A T T T T A T G T T A A T T A A T T T A T T T A C T T T A G T A T T C G G T T T T G T A C C T T T A G C T A T G A T C T T A G C C A T T A T G A T G T T A G A G T T C G C T A T T G G T A T C A T T C A G G G A T A T G T C T G G G C T A T T T T A A C A G C A T C A T A T T T A A A A G A T G C A G T A T A C T T A C A T T A A T T A A T A T T T A C T T A T T A T T A A T A T T T T T A A T T A T T A A A A A T A A T A A T A A T A A T A A T A A T T A T A A T A A T A T T C T T A A A T A T A A T A A A G A T A T A G A T T T A T A T T C T A T T C A A T C A C C T T A T G A A T T C G C

Regulatory sequences of COX2 are underlined, 5’ and 3’-EcoRI sites are in bold italicized characters. The ATP6 sequence is in gray background. 

References

1.         Bonnefoy, N.; Fox, T.D. Genetic transformation of Saccharomyces cerevisiae mitochondria. Methods Cell Biol 2001, 65, 381-396, doi:10.1016/s0091-679x(01)65022-2.

2.         Sherman, F. Getting started with yeast. Methods Enzymol 2002, 350, 3-41, doi:10.1016/s0076-6879(02)50954-x.

3.         Bader, G.; Enkler, L.; Araiso, Y.; Hemmerle, M.; Binko, K.; Baranowska, E.; De Craene, J.O.; Ruer-Laventie, J.; Pieters, J.; Tribouillard-Tanvier, D., et al. Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP. Elife 2020, 9, doi:10.7554/eLife.56649.

4.         Thorsness, P.E.; Fox, T.D. Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA from mitochondria to the nucleus. Genetics 1993, 134, 21-28.

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