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

The method of analyzing protein-protein interactions using the yeast two-hybrid system in Saccharomyces cerevisiae cells is actively used in the search for protein co-regulators. Advantages of this approach include the synthesis of proteins within cells in vivo, the relative speed of this analysis, and high specificity. This method also allows for large-scale screening of libraries of cloned fragments of complementary DNA (cDNA) that are translated within cells. A key factor in the success of such screens is the efficiency of yeast cell transformation, as the resolution of the analysis is based on this. As part of our research, we identified factors that influence the effectiveness of transformation. In particular, we showed that one of the key factors influencing transformation efficiency is the molecular weight of the polyethylene glycol used for chemical transformation of yeast cells and the number of cell division cycles that the cell culture undergoes. We also investigated the effect of the amount and size of plasmids used during transformation. Based on the obtained data, the optimal parameters to achieve a high level of competency in yeast cells were determined. Using an optimized transformation protocol, we screened a cDNA library to explore possible co-regulators of the NIN transcription factor which is a key regulator of the development of the legume-rhizobia symbiosis.

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About the authors

Alina Dymo

All-Russia Research Institute for Agricultural Microbiology

Email: dymoalina@yandex.ru
ORCID iD: 0000-0002-5919-2487
ResearcherId: AAF-3244-2021

младший научный сотрудник

Russian Federation

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

Junior Researcher, Signal Regulation Laboratory

Russian Federation, Saint Petersburg

Alexandra Vyacheslavovna Dolgikh

All-Russia Research Institute for Agricultural Microbiology; Saint Petersburg State University

Email: sqshadol@gmail.com
ORCID iD: 0000-0003-1845-9701
Scopus Author ID: 5719038282
ResearcherId: ABC-2930-2020

engineer

Russian Federation, 3 Podbelsky chausse, Pushkin, Saint Petersburg, 196608; 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

Cand. Sci. (Biol.), Leading Researcher, Laboratory of Molecular and Cellular Biology

Russian Federation, Podbelsky chausse 3, 196608, St.-Petersburg, Russia

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