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. 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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2.
Map plasmid pPICZ-FLP:

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3.
Fig. 1. Scheme for obtaining S. cerevisiae strains with deletions in LEU2 и URA3 genes: a – structure of plasmid pAL2T-delleu2; b – 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) were obtained similarly). Auxotrophic strains to leucine and uracil 3-Y-1236 (Δleu2 Δura3) and 3-Y-2177 (Δleu2 Δura3) were obtained using strains with a single auxotrophy

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4.
Fig. 2. Scheme of M1 toxin fragment integration into the genomes of S. cerevisiae strains and its use as a selection marker: a – structure of plasmid pAL2-T-PGAL1-αTOX; b – obtaining the 4-Y-1236 (LEU2-PGAL1-toxM1 Δura3) and 4-Y-2177 (LEU2-PGAL1-toxM1 Δura3) strains in the genome of which the toxin sequence of the M1 virus was integrated under the control of the regulated promoter of GAL1 gene; c – integration of the 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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5.
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 inhibition zone is the result of the killer toxins action

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6.
Fig. 4. Growth of 4-Y-1236 (LEU2-PGAL1-toxM1 Δura3) and 4-Y-2177 (LEU2-PGAL1-toxM1 Δura3) strains in the genome of which, the M1 virus toxin sequence was integrated under the control of the regulated promoter of the GAL1 gene on media 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 was plated on each agar medium

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7.
Fig. 5. Evaluation of GFP gene integration in transformants genome: a – results of PCR with expaTOX-BHI-F and GFP-R primers and genomic DNA of 1) 4-Y-2177 strain; 3) 4-Y-2177 strain transformed with GFP fragment, clone 1; 4) 4-Y-2177 strain transformed with GFP fragment of clone 2. The sizes of the fragments correspond to the theoretically expected 1343 bp. 2) Evrogen DNA length marker 1 kb; b – scheme representing the location of primers; c – fluorescence microscopy results of the transformed cells in comparison to untransformed 4-Y-2177 strain

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8.
pPICZ-FLP plasmid map:

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

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