Selective system based on fragments of the M1 virus for the yeast Saccharomyces cerevisiae transformation

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Abstract


Background. A selective system based on the M1 virus of the yeast Saccharomyces cerevisiae was proposed.

Methods. To create a recipient strain, a DNA fragment encoding the killer toxin of the M1 virus under the control of the regulated promoter of the GAL1 gene was inserted into the genome of S. cerevisiae strains Y-1236 and Y-2177.

Results. Integration of such expression cassette leads to the conditional lethality – resulting strains die on a medium with galactose when killer toxin synthesis occurs. A linear DNA fragment containing the gene of interest flanked by sequences homologous to the promoter of the GAL1 gene and the termination region of the CYC1 gene is used to transform the obtained strains. During transformation due to homologous recombination, the sequence encoding the killer toxin is cleaved and the transformants grow on a medium with galactose.

Conclusion. The proposed selective system combines the main advantages of other systems: the use of simple media, without the need to add expensive antibiotics, and a simplified technique for constructing expression cassettes and selecting transformants.


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About the authors

Dmitri M. Muzaev

Saint Petersburg State University

Email: dmmuzaev@yandex.ru

Russian Federation, Saint-Petersburg

Engineer

Andrey M. Rumyantsev

Saint Petersburg State University

Email: a.m.rumyantsev@spbu.ru
ORCID iD: 0000-0002-1744-3890
SPIN-code: 9335-1184
Scopus Author ID: 55370658800
ResearcherId: N-3546-2015

Russian Federation, Saint-Petersburg

PhD, Researcher

Ousama R. Al Shanaa

Saint Petersburg State University; Atomic Energy Commission of Syria

Email: oalshanaa@mail.ru

Syrian Arab Republic, Saint-Petersburg; Damascus, Syria

PhD Student

Elena V. Sambuk

Saint Petersburg State University

Author for correspondence.
Email: e.sambuk@spbu.ru
ORCID iD: 0000-0003-0837-0498
SPIN-code: 8281-8020
Scopus Author ID: 6603061322
ResearcherId: H-6895-2013

Russian Federation, Saint-Petersburg

Doctor of Science, Docent

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Supplementary files

Supplementary Files Action
1.
Fig. 1. Scheme for the production of S. cerevisiae yeast strains with deletions in the LEU2 and URA3 genes: a - structure of plasmid pAL2T-delleu2; b - scheme for obtaining the auxotrophic strain 1-Y-1236 (Δleu2) using the FLP-FRT recombination system (strains 2-Y-1236 (Δura3), 1-Y-2177 (Δleu2) and 2-Y-2177 (Δura3) received similarly). Strains with two auxotrophy in leucine and uracil 3-Y-1236 (Δleu2 Δura3) and 3-Y-2177 (Δleu2 Δura3) were obtained using strains with a single auxotrophy as initial

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2.
Fig. 2. Scheme for integrating the sequence of the toxin of the M1 virus into the genome of S. cerevisiae yeast strains and its use as a selective marker: a - structure of plasmid pAL2-T-PGAL1-αTOX; b — scheme for obtaining strains 4-Y-1236 (LEU2-PGAL1-toxM1 Δura3) and 4-Y-2177 (LEU2-PGAL1-toxM1 Δura3), into the genome of which the M1 virus DNA toxin sequence was integrated under the control of the regulated promoter of the GAL1 gene ; c is a diagram of the integration of a GFP gene flanked by sequences homologous to the promoter of the GAL1 gene and the termination region of the CYC1 gene

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3.
Fig. 3. Phenotypes of strains 3-Y-1236 (Δleu2 pYES2-M1) and 3-Y-1236 (Δleu2 pYES2-M28) on a medium with lawns of the control strains 3-Y-1236 (Δleu2 pYES2) and 3-Y-2177 (Δleu2 pYES2). The suppression zone results from the action of killer toxins.

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4.
Fig. 4. The growth of strains 4-Y-1236 (LEU2-PGAL1-toxM1 Δura3) and 4-Y-2177 (LEU2-PGAL1-toxM1 Δura3), into the genomes of which the DNA sequence of the toxin of the M1 virus was integrated under the control of the regulated promoter of the GAL1 gene, on environments with glucose and galactose. The strains are characterized by conditional lethality - they do not grow on media with galactose, since toxin synthesis occurs in their cells under these conditions. 10 μl of a suspension of 104 and 103 cells / ml were sown

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5.
Fig. 5. Checking for the integration of the GFP gene into the transformant genome: a — PCR results with expαTOX-BHI-F and GFP-R primers and genomic DNA strains: 1) 4-Y-2177; 3) 4-Y-2177 with GFP gene integration, clone 1; 4) 4-Y-2177 with the integration of the GFP gene, clone 2. The sizes of the fragments correspond to the theoretically expected 1343 bp; 2) 1 kb DNA length marker (Eurogen). b is an arrangement of primers expαTOX-BHI-F and GFP-R; c - fluorescence of cells of the original strain 4-Y-2177 and the strain transformed with the GFP fragment (clone 2)

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6.
Map plasmid pPICZ-FLP:

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Copyright (c) 2020 Muzaev D.M., Rumyantsev A.M., Al Shanaa O.R., Sambuk E.V.

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