Portal de Periódicos da CAPES

Large-scale yeast transformation in low-percentage agarose medium

2004; Future Science Ltd; Volume: 36; Issue: 1 Linguagem: Inglês

10.2144/04361bm03

1940-9818

Ganna Panasyuk, Ivan Nemazanyy, Valeriy Filonenko, Alexander Zhyvoloup,

Plant tissue culture and regeneration

BioTechniquesVol. 36, No. 1 BenchmarksOpen AccessLarge-scale yeast transformation in low-percentage agarose mediumGanna Panasyuk, Ivan Nemazanyy, Valeriy Filonenko & Alexander ZhyvoloupGanna PanasyukThe Institute of Molecular Biology and Genetics, Kyiv, UkraineNational University "Kyiv-Mohyla Academy", Kyiv, Ukraine, Ivan NemazanyyThe Institute of Molecular Biology and Genetics, Kyiv, Ukraine, Valeriy FilonenkoThe Institute of Molecular Biology and Genetics, Kyiv, Ukraine & Alexander Zhyvoloup*Address correspondence to: Alexander Zhyvoloup, Cell Regulation Laboratory, Ludwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, UK. e-mail: E-mail Address: zhyvoloup@ludwig.ucl.ac.ukThe Institute of Molecular Biology and Genetics, Kyiv, UkraineLudwig Institute for Cancer Research, London, UKPublished Online:6 Jun 2018https://doi.org/10.2144/04361BM03AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The use of yeast as a research tool became especially popular with the development of the yeast two-hybrid (Y2H) assay, which is a powerful tool for the detection of specific protein-protein interactions. Several versions of the Y2H system have been developed. All variations of this method demand the large-scale transformation of yeast with cDNA library plasmids (1). Transformation of yeast with plasmid DNA can be achieved by several methods, such as agitation with glass beads (2), spheroplast preparation (3), treatment with lithium acetate (4), or electroporation (5). The most popular and commonly used is lithium acetate transformation was developed by Ito et al. (6) and modified by Gietz and Woods (7). The standard Y2H library protocols for an average mammalian cDNA library (106 primary independent clones) and maximal colony density 5/mm2 require plating of more than fifty 150-mm plates (8). In addition to being laborious and having problems in maintaining sterility, the inevitable overgrowth of marginal colonies near the plate edge causes loss of uniformity across the library.Here we present a protocol for the large-scale transformation of yeast in a semi-solid agarose medium that does not require plating. The protocol allows for the growth of transformed cells as separate colonies in the volume of semi-solid medium. The protocol is optimized for lithium acetate transformation, gives high transformation efficiency, and provides an equal growth environment for each colony, thereby faithfully preserving the integrity of the primary library. It also allows yeast library propagation with a significantly higher colony density. The protocol was developed on the basis of several published techniques (7–10) and requires fewer reagents when compared to other approaches.Working with DupLEX-A™, a LexA-based Y2H system (OriGene Technologies, Rockville, MD, USA), we successfully used this protocol for the EGY48 strain (MATα, his3, trp1, ura3, 6 LexAop-LEU2) transformed with different pEG202-based bait plasmids and a reporter plasmid pSH18-34 (10). Using this protocol, we managed to achieve transformation efficiencies of 0.25–1 × 106/µg of library DNA or 2–8 million independent colonies/400 mL of the semi-solid agarose medium. Therefore, this protocol allowed us to cover an average cDNA library with just one or two bottles of semi-solid medium (400 mL each).All reagents used were from Sigma (St. Louis, MO, USA) except the low melting point agarose (Invitrogen, Calsbad, CA, USA) and dimethyl sulfoxide (DMSO; VWR, West Chester, PA, USA). Culture media reagents as well as carrier DNA were from Bio101 (Vista, CA, USA). Procedures were performed according to the protocol described below.The plating DOB-His-Ura-Trp medium consisted of Drop Out Base (DOB) medium, which was completed with selective (-His-Ura-Trp) Brent Supplement Mixture (BSM), 2% fructose, and 0.1% low melting point agarose. We have observed that the addition of 2% fructose (but not raffinose or glycerol) helps to lower the background and to facilitate colony visualization in the low melting point agarose medium. All components were mixed on a magnetic stirrer in a final volume of 400 mL of double-distilled water. The solution was microwaved until all components were dissolved and subsequently autoclaved for 7 to 8 min (126°C, 1.7 bar). Alternatively, the medium was prepared from concentrated filter-sterilized stocks, autoclaved 1% agarose, and prewarmed sterile water. The temperature of the agarose medium was adjusted to 40°C in a water bath for 1 h.Transformation of yeast was carried out as described in Table 1 (steps 1–11). Plating medium prepared as described above was mixed with transformed cells by stirring for 1 min and then allowed to solidify on ice for 1 h. The bottle cap was loosened, and growth was continued at 30°C for 36–48 h. Preliminary tests with both aerobically and anaerobically grown yeast showed no difference in recovery rate. Incubation time depended on the density of the inoculated cells and the growth rate. The appearance of microcolonies as a fine-grain structure in the agarose medium was used as the end point for the incubation. The cells were collected as described in Table 1 (steps 12–14).Table 1. Protocol for Large-Scale Yeast Transformation in Low-Percentage AgaroseA small aliquot (1%) of treated cells from step 11 (Table 1) was taken to calculate the efficiency of transformation by the plating assay. Briefly, 10 µL of cell suspension were diluted 100 times with sterile water and spread as follows: 10 µL, 50 µL on yeast peptone dextrose (YPD) plates, and 200 µL, 500 µL on DOB plus BSM-His-Ura-Trp plates. Colonies were counted after 24 h at 30°C.It is important to note that agarose will precipitate together with yeast cells at step 14 of Table 1, which will increase the volume of the pellet up to 100 mL. Therefore, washing and vigorous vortex mixing should be repeated at least three times to release most yeast cells from agarose clogs. The final volume was reduced 10 times by aspirating the excess agarose from above the hard cell pellet. The vast excess of cells allows the loss of some, while aspirating the agarose in order to get a more concentrated library stock.At the final step, a small aliquot of cell suspension was taken for measuring cell density in a cell counting chamber. The information on cell density is very helpful for further Y2H screening. Amplified cells were stored in 25% glycerol (1–2 × 107 cell/mL) at −70°C. The presence of agarose traces has no influence on either the viability of the frozen yeast library stock or the growth in liquid culture and on plates.This protocol has been successfully used in several Y2H screens in our laboratory. A number of positive clones have been isolated by screening mouse embryo libraries (OriGene Technologies) and HeLa and colon tumor libraries (Invitrogen) with bait plasmids for S6K, PTEN, and A33 (Table 2).Table 2. Number of Positive Clones Obtained with Different cDNA Libraries and Bait Plasmids Using Proposed TechniqueThe proposed protocol was validated by comparison to the traditional plating method (10) and showed significant similarities in the pattern of identified binding partners (Table 3). We have found both methods to be equally efficient and that specific growth conditions do not influence colony development at the early log stage. All positives compared were confirmed in a mating assay. Moreover, some of the identified interactions have been confirmed in mammalian cells and published (11–14).Table 3. S6K1 Binding Partner Clones Identified from a Mouse Embryo LibraryAcknowledgmentsThis work was supported in part by grants from The Wellcome Trust and the National Academy of Sciences of Ukraine. G.P. was supported by the European Association for Cancer Research (EACR) Travel Fellowship. I.N. was supported by the International Association for the promotion of cooperation with scientists from the New Independent States of the former Soviet Union (INTAS) Young Scientists Fellowship.References1. Fashena, S.J., I. Serebriiskii, and E.A. Golemis. 2000. The continued evolution of two-hybrid screening approaches in yeast: how to outwit different preys with different baits. Gene 250:1–14.Crossref, Medline, CAS, Google Scholar2. Costanzo, M.C. and T.D. Fox. 1988. Transformation of yeast by agitation with glass beads. Genetics 120:667–670.Crossref, Medline, CAS, Google Scholar3. Hinnen, A., J.B. Hicks, and G.R. Fink. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. USA 75:1929–1933.Crossref, Medline, CAS, Google Scholar4. Gietz, R.D. and R.H. Schiestl. 1991. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier. Yeast 7:253–263.Crossref, Medline, CAS, Google Scholar5. Becker, D.M. and L. Guarente. 1991. High efficiency transformation of yeast by electroporation. Methods Enzymol. 194:182–187.Crossref, Medline, CAS, Google Scholar6. Ito, H., Y. Fukada, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163–168.Crossref, Medline, CAS, Google Scholar7. Gietz, R.D. and R.A. Woods. 2002. Transformation of yeast by the Liac/SS carrier DNA/PEG method. Methods Enzymol. 350:87–96.Crossref, Medline, CAS, Google Scholar8. Clontech Laboratories. 1996. Clontech MATCHMAKER™ LexA Two-Hybrid System User manual. BD Biosciences Clontech, San Jose, CA.Google Scholar9. Invitrogen. 1999. Dual Bait Hybrid Hunter™ Yeast Two Hybrid System Manual. Invitrogen, Carlsbad, CA.Google Scholar10. OriGene Technologies. 1998. DupLEX-A™ Yeast Two-Hybrid System. User's manual, version 2.10. OriGene Technologies, Rockville, MD.Google Scholar11. Balendran, A., R. Currie, C.G. Armstrong, J. Avruch, and D.R. Alessi. 1999. Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252. J. Biol. Chem. 274:37400–37406.Crossref, Medline, CAS, Google Scholar12. Westphal, R.S., R.L. Coffee, Jr., A. Marotta, S.L. Pelech, and B.E. Wadzinski. 1999. Identification of kinase-phosphatase signaling modules composed of p70 S6 kinase-protein phosphatase 2A (PP2A) and p21-activated kinase-PP2A. J. Biol. Chem. 274:687–692.Crossref, Medline, CAS, Google Scholar13. Zhyvoloup, A., I. Nemazanyy, A. Babich, G. Panasyuk, N. Pobigailo, M. Vudmaska, V. Naidenov, O. Kukharenko, et al.. 2002. Molecular cloning of CoA synthase. The missing link in CoA biosynthesis. J. Biol. Chem. 277:22107–22110.Crossref, Medline, CAS, Google Scholar14. Chou, M.M. and J. Blenis. 1996. The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. Cell 85:573–583.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByPI3K/mTOR/S6K signaling pathway – new players and new functional linksBiopolymers and Cell, Vol. 29, No. 3Identification of novel TDRD7 isoformsBiopolymers and Cell, Vol. 27, No. 6Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP413 November 2009 | Molecular and Cellular Biochemistry, Vol. 337, No. 1-2Ribosomal protein S6 kinase 1 interacts with and is ubiquitinated by ubiquitin ligase ROC1Biochemical and Biophysical Research Communications, Vol. 369, No. 2Nuclear Export of S6K1 II Is Regulated by Protein Kinase CK2 Phosphorylation at Ser-17Journal of Biological Chemistry, Vol. 281, No. 42Liquid gel amplification of complex plasmid librariesRebecca Elsaesser & Jacques Paysan6 June 2018 | BioTechniques, Vol. 37, No. 2Coupling homologous recombination with growth selection in yeast: a tool for construction of random DNA sequence librariesClaudia Schaerer-Brodbeck & Alcide Barberis6 June 2018 | BioTechniques, Vol. 37, No. 2 Vol. 36, No. 1 Follow us on social media for the latest updates Metrics Downloaded 265 times History Received 2 September 2003 Accepted 30 October 2003 Published online 6 June 2018 Published in print January 2004 Information© 2004 Author(s)AcknowledgmentsThis work was supported in part by grants from The Wellcome Trust and the National Academy of Sciences of Ukraine. G.P. was supported by the European Association for Cancer Research (EACR) Travel Fellowship. I.N. was supported by the International Association for the promotion of cooperation with scientists from the New Independent States of the former Soviet Union (INTAS) Young Scientists Fellowship.PDF download