Keywords

1 Introduction

The term transformation was first coined by Griffith (1928) describing the conversion of the phenotype of Pneumococcus, from avirulent to virulent. Transformation of Saccharomyces cerevisiae was first accomplished by Hinnen et al. (1978) and Beggs (1978). Ito et al. (1983) first described a method of transforming intact yeast cells utilizing monovalent alkali cations and PEG with a 5-min heat shock at 42°C. Schiestl and Gietz (1989) improved the method by inclusion of single-stranded carrier DNA; however, in 1992 Gietz et al. (1992) showed that the transformation efficiency using the LiAc/ssDNA/PEG method could achieve up to 5 × 106 transformants/μg when certain variables were optimized. Transformation in yeast was reviewed by Gietz and Woods in 2001 (2001) and more recently Kawai et al. (2010).

In the past 12 years much effort has gone into understanding transformation in S. cerevisiae. Hayama et al. (2002) published a method of transformation utilizing only PEG as well as a heat shock or pH jump and call this system “natural transformation.” The levels of transformation with this method were moderate at best; however, this method still required either PEG or 2,3-Dihexadecanoyl-sn-glycero-1-phosphocholine (PCP) and some form of shock to the cells and seemed to work best if the cells were in early log phase growth. These authors suggest that “natural” transformation occurs during early log phase growth and that this system is distinct from the “chemical” method used to achieve highly efficient transformation. Figure 17.1 shows that bar1∆ yeast cells synchronized in G1 phase using α factor and have a peak in transformation efficiency after release from their cell cycle block at early G1/S phase. Once the cells have transitioned to early S phase the transformation efficiency begins to decline. This suggests that a small window of opportunity during the cell cycle is required to obtain highly efficient transformation. Synchronizing a culture should help increase transformation efficiency and yield.

Fig. 17.1
figure 1

Yeast strain DGY233 (MAT a, ade2-1, lys2-1, ura3-52, leu2-3,112, his3∆200, trp1-∆1, bar1-∆1) was grown overnight in YPAD, diluted 1 in 6 into fresh medium, and treated with alpha factor (Sigma) for 5 h. The arrested cells were washed with sterile H2O and resuspended in fresh YPAD and incubated at 30°C. Samples were taken every 15 min and the transformation efficiency of each sample (108 cells) was determined using plasmid YEplac195. Each sample was also examined under a microscope to identify their progression through the cell cycle. T = 120 is M/G1 and T = 135 is early S phase. (Gietz RD, unpublished results)

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Kawai et al. (2004) utilized this natural method of transformation to identify yeast mutants that had altered transformation efficiencies when compared to the parent strain. Approximately 5,000 yeast deletion strains were screened for their ability to transform with this “natural” method. The authors identified a number of mutants with reduced levels of transformation and some mutants with increased levels of transformation. These mutants led the authors to suggest that a type of endocytosis is involved in DNA uptake in S. cerevisiae. We have also screened for mutants that affect transformation utilizing the LiAc/ssDNA/PEG method and our results differ from those presented (Kawai et al. 2004). Table 17.1 shows the mutants, which were identified from a screen of the yeast knock out library purchased from Open Biosystems (Winzeler et al. 1999). In contrast to these authors, we found that sac6 mutants have 0 % transformation in our system. Six other mutants also were shown to have 0 % transformation when tested in our system (Table 17.1). We also found a number of mutants showing an increased level of transformation efficiency when compared to the parent strain BY4742 (Brachmann et al. 1998). The snf12 mutant was shown to have the highest transformation rate of all mutants in our system. In addition five other mutants increased the transformation efficiency from 144 to 324 % of the parental strain. It is clear that these mutants do not overlap with those of Kawai et al. (2004), also listed in Table 17.1, which adds strength to the argument that multiple systems of transformation may be at work in S. cerevisiae.

Table 17.1 Mutants identified screening Open Biosystems yeast deletion collection. Each ORF was replaced with a KanMX antibiotic resistance marker with a unique 20 base pair nucleotide barcode sequence
Full size table

Zheng et al. (2005) first demonstrated that the fluorescent dye YOYO-1 could be used to visualize DNA on the surface of the yeast cell. Li + plus PEG was required to induce 99.4 % of cells to bind YOYO-1 labeled DNA. Later it was shown that Li + treatment affected the topography of the cell wall (Chen et al. 2008). In addition it was found that DNA was only bound to the cell wall when PEG was used. We have shown that plasmid DNA labeled with Alexa Fluor 555 could also be visualized on the yeast cell surfaces (see Fig. 17.2a). In addition, labeled DNA, once bound to the cells by the transformation procedure, remains bound to the cells during vegetative cell growth (Fig. 17.2b). Moreover, this fluorescently labeled DNA that is not taken up by the cell can be removed by a micrococcal nuclease treatment (Fig. 17.2c), leaving a subset of cells where this labeled DNA has been internalized making it refractory to nuclease treatment of an intact cell. Kawai et al. (2004) suggested that during DNA internalization PEG acts on the membrane to enhance the transformation efficiency by increasing the permeability of intact cells. However, PEG treatment caused no intracellular response (Kawai et al. 2009). The model proposes that DNA attaches to the cell wall, passes through and subsequently transits the cell membrane by endocytotic membrane invagination. PEG is suggested to be essential for the DNA attachment to the cell wall. DNA enters the cells via endosomal transport but must escape degradative targeting to the vacuoles and enter the nucleus for the cell to become truly transformed. In addition, visualizing vector DNA with a fluorochrome label to transformation of yeast cells containing a GFP fusion gene showed that it localizes to a specific yeast cell compartment (Huh et al. 2003, Fig. 17.3). This clearly suggests that some labeled internalized DNA seems to co-localize to the endosome compartment labeled with GFP.

Fig. 17.2
figure 2

Yeast transformed with plasmid YEPlac195 labeled with Alexa Fluor 555. Yeast cells were imaged using a Zeiss AxioplanII microscope equipped with a Hamamatus CCD camera. Yeast cells were transformed with 100 ng of plasmid DNA labeled with Alexa Fluor 555 using the high efficiency protocol, fixed with formalin, and then stained with DAPI and visualized using a 63× or 40× lens, (a) yeast cells after standard transformation (630× magnification). (b) Yeast cells transformed with labeled plasmid. Cells were returned to YPAD medium and incubated at 30°C for 48 h with shaking and then a sample of cells was imaged at 630× magnification. (c) Yeast cells transformed with labeled plasmid DNA. Prior to fixation the cells were resuspended in micrococcal nuclease buffer and digested with 5 units of micrococcal nuclease for 15 min at 30°c. The cells were then washed in water, fixed and imaged (400× magnification)

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Fig. 17.3
figure 3

Transformation of endosome GFP strain YLR025W with Alexa Fluor 555 labeled YEPlac195. Yeast cell transformed with labeled plasmid DNA. Prior to fixation the cells were resuspended in micrococcal nuclease buffer and digested with 5 units of micrococcal nuclease for 15 min at 30°c. The cells were then washed in water, fixed and imaged (630× magnification)

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Pham et al. (2011a) showed that the 42°C heat shock was important only to intact cell transformation as spheroplasts did not respond to it. These authors also used negatively charged Nanogold particles in an attempt to study the uptake of DNA into the cell. They observed many Nanogold particles associated with yeast cell wall and membrane as well as some associated with invaginating membranes.

Similar Nanogold particles were used to understand the synergistic effect of LiAc, and ssDNA on transformation efficiency (Pham et al. 2011b). After treatment of cells with LiAc and ssDNA or RbAc and ssDNA, the 42°C heat shock caused the Nanogold particles to be associated with the cell wall, in many cases being trapped inside the cell wall. In addition, these authors suggest that only DNA bound to the cell wall is available for transformation and both ssDNA and LiAc act to modify the yeast cell wall. While much of this model of transformation is reasonable, the role of ssDNA in the transformation reaction is likely nothing more than a quenching agent for the vast number of DNA binding sites that are found on the yeast cell wall after treatment with LiAc and PEG. PEG acts, probably by molecular exclusion, to deposit all large molecular weight DNA and ssDNA and/or RNA onto the surface of the yeast cell wall (Gietz et al. 1995, Gietz and Woods 2001). There are two types of DNA binding sites found on the yeast cell wall: (a) productive, able to give rise to transformants and (b) nonproductive, unable to give rise to transformants. The large quantity of ssDNA floods nonproductive binding sites, allowing plasmid DNA to be more efficiently bound on the productive binding sites and not trapped on nonproductive binding sites, unavailable for transformation. The treatment of cells with LiAc/ssDNA/PEG with a 42°C heat shock causes the structure of cell wall to be altered and DNA bound to the productive DNA binding sites is taken into the cell via endocytosis. Most importantly, the DNA must escape the traditional endosome pathway to allow it to enter the nucleus.

Here, four methods for the transformation of S. cerevisiae are provided. The Quick and Easy transformation method (Gietz and Schiestl 2007a) can be used to give a few hundred transformants when only a few transformants are needed. The High Efficiency Transformation method (Gietz and Schiestl 2007b) can be used to produce millions of transformants. The microtiter plate transformation method can be used for efficient transformation in a 96 well format (Gietz and Schiestl 2007c). Finally, I have included a method for the production of frozen competent yeast cells (Gietz and Schiestl 2007d) that can be prepared in advance and used with high efficiency at a moment’s notice.

2 Materials

2.1 YPAD Medium

YPAD (Yeast Extract-Peptone-Adenine-Dextrose) is used for routine growth of yeast strains prior to transformation as many strains contain the ade2 mutation and grow more vigorously when given adenine. Double strength YPAD broth (2xYPAD) is used to grow cultures to log phase before transformation. Recipes for YPAD and 2xYPAD can be found in Gietz and Woods (2006). These media should be supplemented with adenine hemisulphate at a concentration of 0.1 mg/mL. G418 resistance can be used to select for transformation (Shoemaker et al. 1996).

2.2 SC Selection Medium

SC (Synthetic Complete) selection medium is used for selection of nutritional genetic markers (Gietz and Woods 2006).

2.3 Lithium Acetate (1.0 M)

Dissolve 51.0 g of lithium acetate dihydrate (Sigma Chemical Co. Ltd., St Louis, MO. Catalogue # L-6883) in distilled/deionized H2O and make up to 500 mL, sterilize by autoclave and store at room temperature.

2.4 PEG MW 3350 (50 % w/v)

Add 200 g of PEG 3350 (Sigma Chemical Co. Ltd., Catalogue # P-3640) to 120 mL of ddH2O in a 1 L beaker. Dissolve on a stirring plate. Make the volume up to exactly 400 mL in a graduated cylinder and mix by inversion. Transfer the solution to a storage bottle and autoclave to sterility and stored at room temperature. Ensure your bottle is capped well to prevent evaporation, which will severely affect the yield of transformants.

2.5 Single-Stranded Carrier DNA (2.0 mg/mL)

Dissolve 200 mg of salmon sperm DNA (Sigma Chemical Co. Ltd., Catalogue # D-1626) in 100 mL of TE (10 mM Tris-HCl, 1 mM Na2 EDTA, pH 8.0). You can use a magnetic stir bar over night at 4°C to ensure good dissolution. Aliquots should be stored at –20°C. Carrier DNA should be denatured in a boiling water bath for 5 min and chilled immediately in an ice/water bath before use. Single-stranded carrier DNA can be boiled 3 or 4 times without loss of activity.

2.6 General Equipment

General microbiological supplies are required are listed here. A microtiter plate centrifuge is required for the microtiter plate transformation method. A microtiter plate replicator (Fisher Scientific, Cat# 05-450-9) and a multi-channel micropipettor (Eppendorf™) are also required for the microtiter plate transformation protocols. In addition for the microtiter plate method the plates must be shaken (not stirred) using a rotary shaker. A microtiter plate holder can be fashioned from 1/4 in. plywood or plexiglass by cutting out microtiter plate size rectangles. The plates (plus lids) should fit the slots with minimal play.

3 Methods

3.1 Quick and Easy Transformation Method

  1. 1.

    Inoculate choice of yeast strain onto an YPAD agar plate or in 2 mL of YPAD liquid medium and incubate overnight at 30°C. Footnote 1

  2. 2.

    The following day prepare single-stranded carrier DNA in a boiling water bath for 5 min and chill in ice/water. Footnote 2

  3. 3.

    Scrape a 50 μL blob of yeast cells from the YPAD plate using a sterile loop or toothpick and suspend the cells in 1 mL of sterile water in a microcentrifuge tube. The suspension should contain about 5 × 108 cells. Alternatively, spin down the 2 mL culture and resuspend in 1 mL of sterile water as above.

  4. 4.

    Pellet the cells at top speed in a microcentrifuge for 30 s and discard the supernatant.

  5. 5.

    Add the following components to the pellet in the following order; 1) 240 μL PEG 3500 (50 % w/v), 2) 36 μL lithium acetate 1.0 M, 3) 50 μL SS carrier DNA (2.0 mg/mL), 4) 34 μL plasmid DNA plus dd H2O.Footnote 3

  6. 6.

    Resuspend the cell pellet by briskly vortexing.

  7. 7.

    Incubate the tube in a water bath at 42°C for at least 20 min. Footnote 4

  8. 8.

    Centrifuge the transformation tube at top speed for 30 s and discard the supernatant.

  9. 9.

    Resuspend the cell pellet in 1 mL of sterile water. Use the pipette tip to disrupt the cell pellet, which will aid in resuspension.

  10. 10.

    Plate 200 μL samples of the cell suspension onto five plates with appropriate selection medium. Transformants can usually be isolated after incubation of 3 or 4 days at 30°C.

3.2 High Efficiency Transformation Method

  1. 1.

    High efficiency transformation requires freshly grown yeast cells for best results. Inoculate choice of yeast strain into 5 mL of 2x YPAD or 20 mL of the appropriate selection medium and incubate overnight (16 h) at 30°C on a rotary shaker at 200 rpm. To ensure minimal growth lag pre-warm a culture flask with the medium for the next step.

  2. 2.

    The following day, determine the titer of the yeast culture using one of methods below.

    1. a)

      Dilute 10 μL of culture into a final volume of 1 mL sterile water (1/100 dilution), mix thoroughly and measure the OD at 600 nm (a suspension containing 1 × 106 cells/mL will give an OD600 of about 0.1). Footnote 5

    2. b)

      Dilute 100 μL of culture into a final volume of 1 mL of sterile water (1/10 dilution) in a microcentrifuge tube and mix thoroughly. Deliver 10 μL onto the counting grid of an improved Neubauer hemocytometer, wait several minutes for the cells to settle, and count the number of cells in the 25 large grid squares.Footnote 6

  3. 3.

    Add 2.5 × 108 cells to 50 mL of the pre-warmed 2x YPAD in the pre-warmed culture flask. The titer should be 5 × 106 cells/mL. Alternatively, the titer can be checked after dilution.

  4. 4.

    Incubate the culture in the shaking incubator at 30°C and 200 rpm until the cell titer is at least 2 × 107 cells/mL. This can take about 4 h and at times longer with some strains.

  5. 5.

    Prepare carrier DNA by denaturation in a boiling water bath for 5 min and chill immediately in an ice/water bath. Footnote 7

  6. 6.

    Harvest the cultured cells once they have reached the correct titer by centrifugation at 3,000 g for 5 min, wash twice with 25 mL of sterile water and resuspend the cells in 1 mL of sterile H2O.

  7. 7.

    Pellet the cells in a fresh 1.5 mL microcentrifuge tube by centrifugation at maximum speed for 30 s and discard the supernatant.

  8. 8.

    Resuspend the cell pellet in 500 μL of sterile ddH2O and transfer 50 μL samples containing 108 cells into fresh 1.5 mL microcentrifuge tubes for each transformation. Pellet cells at top speed for 30 s in a microcentrifuge and remove the supernatant.

  9. 9.

    Add the Transformation Mix to each tube containing cell pellet and resuspend the cells by vigorous vortexing. Transformation mix is made up prior to this step and stored on ice and the following components added in the following order and then mixed on a vortex mixer: 1) PEG 3500 (50 % w/v) 240 μL, 2) lithium acetate 1.0 M 36 μL, 3) SS carrier DNA (2.0 mg/mL) 50 μL, 4) plasmid DNA and sterile ddH2O 34 μL. Make additional aliquots calculating the number of transformations planned.

  10. 10.

    Place the tubes in a 42°C water bath for 40 min.Footnote 8

  11. 11.

    Pellet the cells in a microcentrifuge at top speed for 30 s and remove the Transformation Mix. Use a pipettor to remove as much of the Transformation mix as possible.

  12. 12.

    Resuspend the cell pellet in 1 mL of sterile ddH2O. This can be difficult therefore add a small amount of sterile ddH2O and stir the pellet with a micropipette tip to aid in suspension of the cells followed by vigorous vortexing.

  13. 13.

    Plate the resuspended cells onto the appropriate selection medium. A good transforming strain will give up to 2 × 106 transformants/μg plasmid DNA/108 yeast cells. Plate 2, 20, and 200 μL onto the appropriate selection medium. Footnote 9

  14. 14.

    Incubate the plates at 30°C for 3–4 days to recover transformants.

This method can be used to generate large numbers of transformants required to screen complex clone libraries such as a two-hybrid or similar screens. It is advisable to test the effects of increasing plasmid DNA on transformation efficiency before embarking on a large screen. This information will allow the determination of the appropriate scale up factor (30×, 60×, or 120×) and the appropriate plasmid amount to obtain the number of transformants required to cover the DNA library complexity with high probability. Specific considerations for these screens are found in Gietz (2006).Footnote 10, Footnote 11, Footnote 12

3.3 Microtiter Plate Transformation Method

A method for the transformation of yeast cells in 96 well microtiter plate format is presented here. Sterile 96 well microtiter plates with round bottoms and lids are used for this method. The Microtiter Plate Protocols can be adapted for a number of purposes. A) Many different yeast strains can be grown on a master plate, sampled with a replicator into the wells of a microtiter plate and tested for transformation efficiency with a single plasmid. B) A single strain can be transformed with many different plasmids (e.g., a plasmid library in a 96 well format). C) Many yeast strains can be grown on a master plate, transferred to wells containing 150 μL of 2xYPAD, re-grown in sealed plates on a shaker at 200 rpm, and then transformed in situ with a single plasmid. A 96-prong replicator and 150 mm petri dishes of medium are used for this method. The Transformation Mix for these protocols is prepared without the PEG reagent making the cell pellet easier to resuspend. The PEG is added after the cell pellets have been resuspended. This method can use an agar plate method or a liquid method for growth of the cells to be transformed depending on your specifications.

3.3.1 Agar Plate Method

  1. 1.

    The 96 well replicator prongs are sterilized by dipping in a petri plate containing 70 % ethanol and then passing it through a Bunsen flame and cooling. Footnote 13

  2. 2.

    Carefully set the cooled prongs of the sterile replicator onto the surface of a 150 mm YPAD plate. This will print the position of each well on the agar plate.

  3. 3.

    Patch the yeast strain(s) onto the positions as necessary. Be sure to mark the orientation of the master plate and incubate overnight at 30°C.

  4. 4.

    The following day pipette 150 μL samples of sterile water into each well of the microtiter plate.

  5. 5.

    Denature carrier DNA (2 mg/mL) for 5 min in a boiling water bath and chill in ice/water.

  6. 6.

    Cool the sterilized replicator by dipping prongs into microtiter plate containing sterile water.

  7. 7.

    Place the prongs of the cooled replicator onto the plate, making sure that each prong contacts a patch of yeast inoculum. Gently move the replicator in small circles to transfer cells to the prongs taking care not to cut into the agar surface. Remove the replicator and inspect the prongs for yeast cell coverage.

  8. 8.

    Place the replicator into the microtiter plate containing the sterile water and agitate in a circular motion to wash the cells off the replicator prongs. This will give approximately 1 × 107 cells per well. Repeating the transfer process will increase the number of cells, if necessary. Mark the orientation of the microtiter plate.

  9. 9.

    Pellet the cells by centrifugation 10 min at 1,300 g using a microtiter plate rotor.

  10. 10.

    Remove the supernatant from the wells. This may be accomplished by aspiration or dumping in a sink followed by a sharp flick to remove the last remaining drop. This technique should be practiced prior to using it on a screen.

  11. 11.

    Mix the Microtiter plate Transformation Mix as indicated in Table 17.2. The volumes listed are for a single transformation (each well). Make sufficient for 100 transformations if you intend to use all 96 wells. The plasmid amount can be increased but the volume must stay the same.

    Table 17.2 Microtiter plate transformation mix volumes
    Full size table
  12. 12.

    Deliver 50 μL of the Microtiter plate Transformation Mix into each well. Secure the microtiter plate to a rotary shaker at 400 rpm for 2 min to resuspend the cell pellets.

  13. 13.

    Pipette 100 μL PEG 3350 (50 % w/v) into each well and place back onto rotary shaker for 5 min at 400 rpm to mix the PEG and cell suspensions.

  14. 14.

    Place each microtiter plate into plastic bag or seal with Parafilm™ and incubate at 42°C for 1–4 h. Footnote 14

  15. 15.

    Centrifuge each microtiter plate for 10 min at 1,300 g, remove the supernatant by aspiration, and resuspend the cells by adding 50 μL of sterile water to each well followed by placement on a rotary shaker at 400 rpm for 2–5 min. Microtiter plate wells may be sampled individually by sampling a 5 μL aliquot from a well into 100 μL puddles of sterile water on plates of selection medium. A sample can be taken using the replicator to print samples (ca; 5–10 μL) onto selection plates. Multiple samples can be delivered onto large plates using the replicator if care is used to print samples to the exact positions. Incubate the plates at 30°C for 2–4 days and recover the transformants.

3.3.2 Liquid Culture Protocol

The yeast cells of the re-grown culture are harvested, washed, and resuspended in water and the cell titer determined as described earlier (17.3.2.2.).

  1. 1.

    Adjust the titer of the cell suspension to 5 × 108 cells/mL and dispense 100 μL of the suspension into the wells of the microtiter plate.

  2. 2.

    Continue from step 9 of the agar plate protocol but increase the amount of plasmid to 100 ng/transformation.

  3. 3.

    Seal and incubate the plates at 42°C for 60 min.

  4. 4.

    Sample the wells using a pipette or microtiter replicator onto selection medium.

  5. 5.

    Incubate the plates at 30°C for 2–4 days and recover and/or count the transformants.

3.4 Transformation-competent Frozen Yeast Cells

This method can be used to produce frozen competent yeast cells when a single yeast strain is used repeatedly. Yeast cultures are re-grown for at least two divisions and used to produce transformation-competent cells that are frozen and used at a moment’s notice.

3.4.1 Preparation

  1. 1.

    The yeast strain is grown overnight and then re-grown in 2x YPAD to a titer of 2 × 107 cells/mL as described in 17.3.2. One hundred samples of 1 × 108 frozen competent cells will require 500 mL of re-grown culture (1 × 1010 cells).

  2. 2.

    Harvest the cells by centrifugation at 3,000 g for 5 min, wash the cells in 0.5 volumes of sterile water, and resuspend in 5 mL of sterile water. Transfer to a suitable sterile centrifuge tube and pellet the cells at 3,000 g for 5 min.

  3. 3.

    Resuspend the cell pellet in 5 mL of frozen competent cell (FCC) solution (5 % v/v glycerol, 10 % v/v DMSO). Use high quality DMSO for best results.

  4. 4.

    Dispense 50 μL samples into an appropriate number of 1.5 mL microfuge tubes.

  5. 5.

    Place the microfuge tubes into a 100 tube Styrofoam rack with lid (Sarstedt #95.064.249) or similar type of rack. It is best to place this container upright in a larger box (Styrofoam or cardboard) with additional insulation such as foam chips or newspaper to reduce the air space around the samples. This will result in the samples freezing slowly, which is essential for high survival rates.

  6. 6.

    Place the container at –80 °C overnight. The tubes can then be removed from the tube rack container and stored at –80°C in bulk. These cells can be stored for up to 1 year with little loss of transformation efficiency.

3.4.2 Transformation of Frozen Competent Yeast Cells

These cells are transformed using a modified High Efficiency Transformation method 3.2 with the differences listed below.

  1. 1.

    Thaw cells in a 42°C water bath for 15 s.

  2. 2.

    Pellet the cells at 10,000 g in a microcentrifuge for 2 min and remove the supernatant.

  3. 3.

    Add 360 μL of FCC transformation mix (260 μL 50 % PEG, 36 μL 1.0 M LiOAc, 50 μL denatured carrier DNA, and 14 μL of DNA and water) and vortex vigorously to resuspend the cell pellet. Note the difference in PEG volume.

  4. 4.

    Incubate in a 42°C water bath for 20–60 min depending on the strain. Centrifuge as above to remove the supernatant and resuspend the cell pellet in 1 mL of sterile water.

  5. 5.

    Plate appropriate dilutions onto selection medium.