Optimization of transformation conditions of the yeast Saccharomyces cerevisiae to determine coregulators of the transcription factor NIN in a yeast two-hybrid system

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Abstract

BACKGROUND: The method of protein-protein interaction analysis using the yeast two-hybrid system in Saccharomyces cerevisiae cells is used to search for protein coregulators. This method is also used for mass screening of libraries of cloned fragments of complementary DNA (cDNA) that are translated in the cell. The key factor in the success of such screening is the level of efficiency of yeast cell transformation, since the resolution of the analysis is based on this.

AIM: The aim of this study is to search for optimal parameters for chemical transformation of yeast cells to increase the resolution of cDNA library screening.

MATERIALS AND METHODS: Plasmids pDEST22 and pDEST32 were used for chemical transformation of the yeast strain S. cerevisiae pJ69-4A. For screening, a cDNA library was used, obtained on the basis of mRNA isolated from the roots of pea Pisum sativum cultivar Finale, inoculated with rhizobia.

RESULTS: Factors influencing the efficiency of transformation were identified. Among them are the molecular weight of polyethyleneglycol used for transformation, as well as the number of cell division cycles that the culture undergoes. The effect of the number and size of plasmids used in transformation was also shown. Using the optimized protocol, a cDNA library was successfully screened to find coregulators of the NIN transcription factor.

CONCLUSIONS: Based on the data obtained, optimal parameters were determined that allow achieving a high level of competence in yeast cells. The use of the described protocol allowed for successful screening of the library to identify coregulators of the NIN transcription factor.

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About the authors

Alina M. Dymo

All-Russia Research Institute for Agricultural Microbiology

Email: dymoalina@yandex.ru
ORCID iD: 0000-0002-5919-2487
ResearcherId: AAF-3244-2021
Russian Federation, Saint Petersburg

Elizaveta S. Kantsurova

All-Russia Research Institute for Agricultural Microbiology

Email: rudaya.s.e@gmail.com
ORCID iD: 0000-0002-3081-9880
SPIN-code: 4752-1910
Russian Federation, Saint Petersburg

Alexandra V. Dolgikh

All-Russia Research Institute for Agricultural Microbiology

Email: sqshadol@gmail.com
ORCID iD: 0000-0003-1845-9701
SPIN-code: 2602-1514
Scopus Author ID: 5719038282
ResearcherId: ABC-2930-2020
Russian Federation, Saint Petersburg

Elena A. Dolgikh

All-Russia Research Institute for Agricultural Microbiology

Author for correspondence.
Email: dol2helen@yahoo.com
ORCID iD: 0000-0002-5375-0943
SPIN-code: 4453-2060
Scopus Author ID: 6603496335
ResearcherId: G-6363-2017

Dr. Sci. (Biology)

Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. Scheme of screening of protein-synthesizing library in yeast two-hybrid system. 1 — Cloning of cDNA pool into pDEST22 vector; 2 — transformation of S. cerevisiae yeast cells with pDEST32 plasmid containing coding sequence of NIN gene of pea P. sativum; 3 — plating of cells after transformation on selective medium without leucine to select colonies containing pDEST32 plasmid with leucine biosynthesis gene LEU2; 4 — preparation of competent yeast cells containing pDEST32 plasmid; 5 — transformation of cells containing pDEST32 plasmid with pDEST22 plasmid, for which selection is carried out on medium without tryptophan; 6 — plating of cells after transformation on a selective medium without leucine, tryptophan and histidine, as well as a medium with the addition of 3-aminotriazole to select colonies containing plasmids pDEST22 and pDEST32 and showing the possibility of interaction of the synthesized proteins

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3. Fig. 2. Evaluation of the effect of the medium used for growing the yeast culture (a) and the number of division cycles the culture undergoes during growth on the efficiency of yeast cell transformation (b). The statistical significance of the observed differences was assessed using the Wilcoxon test (Mann–Whitney test) (a) and one-way ANOVA with Tukey’s post-hoc test (b). Different letters (a, b) indicate statistically different values (p < 0.05) based on the post-hoc test data. The Shapiro–Wilk test was used to assess the normality of data distribution

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4. Fig. 3. Determination of the optimal incubation time at elevated temperature (a) and the effect of such components of the transformation mixture as PEG with average molecular weights of 3350, 4000, 6000, and 20000 (b) and ballast single-stranded DNA in an amount of 50, 100, and 200 μg (c) on the transformation efficiency of the yeast strain pJ69-4A. One-Way ANOVA with Tukey’s post-hoc test (a, b) and Kruskal–Wallis one-way analysis (c) were used to assess statistical significance, and the Shapiro–Wilk test was used to assess the normality of data distribution. Different letters (a, b) indicate statistically different values (p < 0.05) based on the post-hoc test data, and double letters (ab) indicate that the values in this group are not statistically different from a and b but different from c

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5. Fig. 4. Evaluation of the efficiency of yeast cell transformation using plasmids of different sizes pDEST22 (8.9 kb) and pGBT9 (5.5 kb) with the tryptophan biosynthesis gene TRP1. The statistical significance of the observed differences was assessed using the Wilcoxon T-test (Mann–Whitney test). The Shapiro–Wilk test was used to assess the normality of data distribution

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6. Fig. 5. Results of PCR analysis of plasmids isolated from 10 transformants carrying empty pDEST32 plasmid and pDEST22-based library plasmids with different inserts. The PCR reaction used primers flanking the insert in pDEST22 plasmid

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