Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

1. UV Mutagenesis to Generate Auxotrophic Yeast Strains

1. Generate survival curves to determine needed doses of UV irradiation
   1. Prepare YPD (yeast extract peptone dextrose) media and other reagents listed in Table 1 according to standard procedures²⁶ and inoculate single colonies into 5-10 ml of YPD media. Incubate cultures on a roller drum at 30 °C O/N to saturation for at least 8 hr.
   2. Determine the cell concentration of O/N cultures using a spectrophotometer by diluting cells 1:10 in water in a plastic cuvette. Dilute cells to a concentration of 10⁷ cells/ml in 20 ml sterile distilled water.
   3. Pour diluted cells into a sterile plastic Petri dish and, with the lid removed, place the plate 14 cm below a UV bulb.
   4. Expose cells to serial 5,000 µJ and 10,000 µJ doses of UV irradiation, extracting 500 µl of cells following each increment such that cells are sampled after exposure to 0 µJ, 5,000 µJ, 10,000 µJ, 15,000 µJ, 20,000 µJ, 25,000 µJ, 30,000 µJ, 40,000 µJ, and 50,000 µJ of UV irradiation. Transfer extracted cell samples to sterile 1.5 ml tubes and serially dilute at 1:10 increments in sterile water.
   5. Pellet cells in each dilution by centrifugation in a microcentrifuge at 16,000 x g for 1 min. Aspirate supernatant and resuspend in a 100 µl volume of sterile water appropriate for plating yeast cells. Pipette the full volume of resuspended cells onto plates containing YPD solid media and use a sterile spreader to evenly distribute cells across each plate.
   6. Wrap plate edges in Parafilm to prevent drying of media and cover plates in aluminum foil to prevent photo-reactivation and repair of UV-induced mutations. Incubate plates upside down at 30 °C for 2-4 days to allow for growth of viable yeast colonies (Figure 1).
   7. Count the number of colonies, optionally with the help of a pen to mark off counted colonies, a hand held electronic counter pen, or a counter stand with magnification. Plot as a percentage of total plated cells at each µJ dose of UV irradiation to generate a survival curve for irradiated yeast (Figure 2).
      NOTE: Haploid yeast strains can be expected to require lower doses of UV irradiation relative to diploid strains to reach the same percent survival. A strain of yeast lacking functional DNA repair enzymes, such as the rad1 S. cerevisiae mutant, can be used as a positive control to indicate the presence of UV irradiation at very low doses.
   8. Determine the dose of UV mutagenesis to be used for screening by referring to the survival curve established in 1.1.7. The x value of the point along the survival curve where y equals 50 is the UV irradiation dose at which 50% of yeast survive. Screening mutants at this low percent survival may result in a higher yield of successfully mutated strains, particularly for diploid yeast. The 50% survival dose for WT S. boulardii, as shown in Figure 2, is approximately 18,000 µJ.
      NOTE: Although such high UV doses increase the risk of mutations in genes for cellular pathways other than the auxotrophic marker gene of interest, this drawback must be balanced against the need to induce mutations in both copies of the auxotrophic marker gene. For haploid strains in which only one gene copy must be mutated, screening at a higher percent survival, such as at 90%, decreases the risk of additional mutations and still allows for sufficient generation of auxotrophic mutants.
2. UV mutagenesis and screening for auxotrophic yeast strains
   1. Prepare yeast as described in steps 1.1.1-1.1.3.
   2. Expose yeast to the dose of UV irradiation corresponding to 50% survival, as determined in 1.1.8. For WT S. boulardii, this dose was determined to be approximately 18,000 µJ (Figure 2).
   3. Collect 1 ml volumes of UV irradiated yeast and pellet by centrifugation in a microcentrifuge at 16,000 x g for 1 min. Aspirate supernatant and resuspend cells in 100 µl sterile water.
      1. Selection of mutants
         1. If using selection such as with ura3^– auxotrophic mutants, plate irradiated yeast onto media containing 5-fluoroorotic acid (5-FOA) to select for cells lacking a functional Ura3 enzyme.
            NOTE: Any yeast containing functional copies of Ura3 will convert 5-FOA to the toxin 5-fluorouracil, leading to cell death and allowing for easy selection of ura3–colonies that lack a functional Ura3²⁷. Analogous selection approaches are possible for the LYS2 and LYS5; TRP1; and MET2 and MET15 markers using media containing α-aminoadipic acid²⁸; 5-fluoroanthranilic acid²⁹; and methyl mercury^30,31, respectively.
         2. Pipette the 100 µl of resuspended cells onto minimal media containing 5-FOA and use a sterile spreader to evenly coat the plate. Wrap plates in Parafilm and incubate upside down at 30 °C for 2-4 days to allow for growth of viable yeast colonies.
         3. Confirm the ura3^– phenotype of any colony appearing on 5-FOA plates by restreaking onto YPD, uracil^– and 5-FOA plates (Figure 3). Use the tip of a sterile toothpick to collect part of a single colony and gently drag the cells across fresh YPD, uracil^–, and 5-FOA plates. Again incubate wrapped plates upside down at 30 °C for 2-4 days.
            NOTE: Viable colonies will appear as raised, roughly circular growths while non viable cells will appear only as an opaque smear without any raised growths (Figure 3).
      2. Screening of mutants
         1. If generating an auxotrophic mutant for which selection methods are not available, prepare serial 1:10 dilutions of UV irradiated yeast cells in sterile water and pipette the dilutions onto YPD media, using a sterile spreader to evenly coat the plate.
         2. Wrap plates in Parafilm and incubate upside down at 30 °C for 2-4 days. Determine which dilution allowed for growth of individual colonies that can easily be distinguished from each other, usually no more than approximately 100 colonies per plate.
         3. Repeat UV mutagenesis of yeast samples as described in 1.2.1-1.2.3 and plate cells at this determined dilution. Pipette the diluted yeast onto YPD media, use a sterile spreader to distribute the cells, and incubate wrapped plates upside down at 30 °C for 2-4 days.
         4. Screen for auxotrophs by replica plating onto selective media lacking the metabolite of interest. First, secure a sterile velvet pad onto a plate stand and invert the plate with UV irradiated colonies onto the velvet, marking the plate orientation.
         5. Next, invert a fresh plate lacking the metabolite of interest onto the velvet and lightly press down to transfer cells from the velvet to the plate. Store the original plate at 4 °C. Wrap and incubate the new plate upside down at 30 °C for 2-4 days.
         6. As an alternative to replica plating, screen mutants by re-streaking colonies from YPD onto selective media. Use the tip of a sterile toothpick to collect part of a single colony and gently drag the cells across a fresh YPD plate and a plate lacking the metabolite of interest. Again incubate wrapped plates upside down at 30 °C for 2-4 days.
            NOTE: Care must be taken to select single colonies and streak out colonies multiple times to confirm a homogeneous population of true auxotrophic cells.
   4. Further confirm the phenotype of irradiated cells by inoculating single colonies into 5-10 ml of both YPD and media lacking the appropriate metabolite (e.g., in uracil^– media for a ura3^– mutant). Incubate on a roller drum at 30 °C O/N to confirm growth of cells in YPD media but not in the absence of the metabolite.
      NOTE: Although growth patterns on solid media should clearly indicate yeast auxotrophic status, it is possible for some yeast to tolerate stresses and form small, slow growing colonies on solid media but yet not tolerate the same conditions in liquid media (unpublished observations). Inoculation into liquid cultures should thus be performed to thoroughly confirm growth patterns of UV irradiated mutants.
   5. For long term storage of confirmed auxotrophic mutants, prepare glycerol stocks by inoculating cells into 10 ml YPD and incubating on a roller drum O/N at 30 °C. Pellet cells by centrifugation for 3 min at 2,500 x g and aspirate media. Resuspend cells in 50% sterile filtered glycerol, transfer to a cryovial, and store at -80 °C.
      NOTE: UV mutagenized yeast potentially contain mutations in multiple genes other than in the auxotrophic marker of interest. Before continuing with use of verified auxotrophic mutants, these strains should be further analyzed through gene sequencing and assessment of resistance to pH, bile acid stresses, and other characteristics relevant to probiotic strains, as described elsewhere². Additionally, use of pcr homology or CRISPR/Cas9 targeting to more selectively mutate auxotrophic markers should be considered as an alternative to UV mutagenesis^4–6.

2. Yeast Transformation

1. LiOAc Transformation of Yeast
   1. Inoculate single yeast colonies into 5-10 ml of YPD media and incubate on a roller drum at 30°C O/N.
   2. To induce log phase growth and increase efficiency of plasmid uptake, determine cell concentration using a spectrophotometer to measure a 1:10 dilution of cells in sterile water in a plastic cuvette. Dilute O/N cultures to an OD₆₀₀ of 0.16-0.2 (approximately 2 x 10⁶– 2.5 x 10⁶ cells/ml) in 50 ml of fresh warm YPD and incubate cells on an orbital platform shaker set to 200 rpm until the culture reaches approximately 1 x 10⁷ cells/ml, usually around 4 hr.
      NOTE: Transformation efficiency can be measured as a function of the number of successfully transformed yeast colony forming units (CFU) per µg of plasmid DNA. Increased efficiency results in more transformed colonies per µg of plasmid DNA. Subculturing yeast cells and collection during log phase growth is one factor that increases transformation efficiency¹².
   3. Pellet cells by centrifugation at 2,500 x g for 3 min.
   4. Aspirate the supernatant and transfer cells to a 1.5 ml microcentrifuge tube by resuspending the pellet in 1 ml sterile water.
   5. Pellet cells by centrifugation at 16,000 x g for 1 min in a microcentrifuge, aspirate the supernatant and wash cells by resuspension in 1 ml TE/LiOAc (Tris EDTA LiOAc) buffer.
   6. Repeat centrifugation and resuspend cells in TE/LiOAc buffer to a concentration of 2 x 10⁹ cells/ml.
   7. Prepare transformation mixtures with each of the following: 50 µl of prepared yeast in TE/LiOAc buffer, 5 µl of carrier DNA (10 µg/µl), and 1 µl of plasmid DNA (1 µg). Micrograms of plasmid DNA may be titrated as increasing amounts of DNA may or may not lead to increased transformation efficiency³²
      NOTE: For a mutant yeast strain lacking one auxotrophic marker, only one plasmid encoding the marker can be transformed per sample. Furthermore, use of a plasmid encoding an easily detectable protein, such as GFP, will allow for efficient determination of proper folding and expression of heterologous protein by the yeast strain subsequent to transformation.
   8. To each preparation, add 300 µl of PEG/LiOAc/TE and vortex thoroughly. Incubation of intact cells with PEG is essential for efficient transformation¹³.
   9. Incubate preparations at 30 °C for 30 min with agitation by placing microcentrifuge tubes in a beaker placed onto an orbital platform shaker at 200 rpm.
   10. Add 35 µl of DMSO to each reaction and heat shock cells for 15 min in a 42 °C water bath. Although there are conflicting reports of the added benefit of DMSO³³, heat shock of intact yeast cells has been shown to greatly increase transformation efficiency¹².
   11. Wash cells by pelleting via centrifugation as in 2.1.5, aspirating or pipetting off the supernatant, and resuspending in 1 ml sterile water. Gently pipette up and down to break up the cell pellet.
       NOTE: It is critical to thoroughly remove the supernatant because the media used in generating competent cells can inhibit yeast growth and colony formation.
   12. Repeat cell pelleting as in 2.1.5, aspirate the supernatant, and resuspend the cells in 100 µl sterile water. Pipette the full volume onto a selective plate and use a sterile spreader to evenly coat the plate with transformed yeast.
   13. Wrap edges of coated plates in Parafilm to prevent drying of media and incubate upside down at 30 °C for 2 days to allow for growth of transformed yeast cells. Successful, efficient transformation and auxotrophic selection of Saccharomyces cerevisiae yields a high number of colonies per transformation preparation, although yield can be much lower for other strains (Figure 4).
   14. Store yeast plates for the short term (generally 1-3 weeks) upside down and covered at 4 °C. Prepare glycerol stocks of transformed yeast as described in 1.2.5 for long term storage.
       NOTE: Further studies testing transformed CFU are necessary to determine plasmid stability and evaluate proper expression of heterologous protein. Thorough descriptions of plasmid stability³⁴, use of immunoblotting to detect denatured proteins recovered from cell samples¹⁷, enzyme linked immunosorbent assay (ELISA) to detect properly folded three dimensional proteins ¹⁶, and the use of GFP in yeast studies³⁵ are available elsewhere. Figure 6 shows a representative image of successful GFP expression in transformed S. cerevisiae relative to untransformed cells. Use of such a fluorescent protein is one means of efficiently determining successful heterologous protein production.
2. Electroporation of Yeast
   1. Inoculate single yeast colonies into 5-10 ml of YPD media and incubate on a roller drum at 30 °C O/N.
   2. Determine cell concentration using a spectrophotometer to measure a 1:10 dilution of cells in sterile water. Dilute O/N cultures in 100 ml fresh warm YPD media to an OD₆₀₀ equivalent of approximately 0.3. Incubate subcultures at 30 °C on an orbital platform shaker set to 200 rpm until reaching an OD₆₀₀ of approximately 1.6, usually 4-5 hr. Each 100 ml subculture will generate enough conditioned cells for two transformation reactions.
   3. Pellet cells by centrifugation at 2,500 x g for 3 min. Aspirate the supernatant and wash cells by resuspending in 50 ml ice cold sterile water. Repeat the wash by pelleting cells, aspirating supernatant, and resuspending in fresh 50 ml ice cold sterile water.
   4. Pellet the cells again and resuspend in 50 ml ice cold electroporation buffer (1 M Sorbitol, 1 mM CaCl₂).
   5. Repeat spin as in 2.2.3, aspirate supernatant, and resuspend cells in 20 ml 0.1 M LiOAc/10 mM DTT. Incubate cell suspension on a roller drum at 30 °C for 30 min. Preincubation of cells in LiOAc and DTT synergistically increases the efficiency of electroporation³⁶.
   6. Pellet the cells as in 2.2.3, remove supernatant, and wash by resuspending in 50 ml ice cold electroporation buffer. Repeat centrifugation and resuspend cells in ice cold electroporation buffer to a final volume of 1 ml.
   7. Prepare on ice: conditioned yeast cells, sterile electroporation cuvettes, and plasmid DNA. Immediately after final resuspension of conditioned cells in 1 ml electroporation buffer, combine 400 µl conditioned yeast cells with approximately 1 µg of plasmid DNA and add to an ice cold 0.2 µm electroporation cuvette. Use of increased amounts of DNA may slightly increase transformation efficiency¹¹. Incubate reaction on ice for 5 min, then electroporate with electroporator set to 2.5 kV and 25 µF.
      NOTE: As described in 2.1.7, a mutant yeast strain lacking one auxotrophic marker can be transformed with only one plasmid encoding the mutated marker per sample. Also, using a plasmid that encodes an easily detectable protein such as GFP allows for efficient determination of proper folding and expression of heterologous protein subsequent to transformation.
   8. Transfer electroporated cells into 8 ml of a 1:1 mixture of YPD:1 M sorbitol and allow cells to incubate on a roller drum at 30 °C for 60 min.
   9. Pellet cells as in 2.2.3 and resuspend in 100 µl 1:1 YPD:1 M sorbitol. Plate the full volume onto selective media containing 1 M sorbitol, wrap edges of plates in Parafilm, and incubate plates upside down at 30 °C for 2-5 days to allow for growth of transformed yeast cells.
      NOTE: It is critical to test transformed yeast to evaluate proper expression of heterologous protein, as described in 2.1.14 and 2.2.7.

3. Oral Gavage of Mice with Transformed Yeast

1. Perform all animal care and handling procedures according to the Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee approval.
2. Prepare O/N yeast cultures by inoculating single colonies of transformed auxotrophic yeast into 5-10 ml of selective media. Incubate cultures O/N for at least 8 hr on a roller drum at 30 °C until saturated.
   NOTE: Use of plasmid encoding test proteins such as GFP will allow for ease of protein expression testing in gavaged yeast, as described in 4.7.
3. For maximal induction of protein expression and to induce log phase growth, prepare subcultures from the O/N cultures by diluting to an OD₆₀₀ equivalent of approximately 0.16-0.2 in 50 ml of appropriate media as described in 2.1.2.
4. Determine the concentration of subcultured cells as in 1.1.2 and adjust to 10⁹ cells/ml. Prepare a 100 µl dose for each mouse, with a few hundred µl extra volume per group to improve accuracy and ease of sample loading.
5. Pellet cells by centrifugation at 2,500 x g for 3 min or in a microcentrifuge at 16,000 x g for 1 min. Aspirate supernatant and resuspend cells by adding an equal volume of sterile water and gently pipetting up and down.
6. Fix an appropriate gauge gavage needle (22 G for 15-20 g mice) onto a 1 ml sterile syringe and load yeast sample, being sure to eliminate any bubbles and set plunger to a 100 µl increment. Load an additional syringe with sterile water to gavage control mice and check for presence of any contaminating yeast.
7. Pick up the mouse to be gavaged using the non-dominant hand, with index finger and thumb tightly grasping the skin around the neck (Figure 6A). Tuck the tail under the small finger to prevent movement of the lower body. Be sure that the grip is secure and prohibits the mouse from moving its head in order to prevent damage to internal tissues during gavage.
   NOTE: Estimate how far the gavage needle should be inserted by holding the needle against the mouse such that the bulb is even with the xiphoid process of the sternum. Inserting this length of the needle will typically allow the gavage needle bulb to enter the stomach.
8. Using the dominant hand, gently insert the gavage needle into the mouse esophagus by angling the needle along the roof of the mouth and back of the throat, keeping slightly to the left of center. Wait for the mouse to swallow the bulb of the needle and allow the needle to descend slightly further to the point estimated in 3.7 (Figure 6B). If any resistance is felt during insertion of the gavage needle or if the mouse at any time begins to gasp, gently remove the needle and again try to find the esophagus.
9. After the mouse has swallowed the bulb of the gavage needle, gently depress the syringe plunger to administer 100 µl (10⁸ CFU) of yeast directly into the mouse stomach.
   NOTE: Although mice are unable to vomit any of the gavaged solution after administration^18,19, it is possible for reflux to occur during gavage^21,37. Proper insertion of the gavage needle, as well as adjusting the volume and viscosity of the solution, can help to limit reflux and ensure accurate dosing.
10. Carefully remove the gavage needle from the mouse stomach and esophagus and return the mouse to the cage. Check that the mouse is breathing and moving normally after gavage to ensure that the gavage needle was properly inserted throughout the procedure and that no solution was aspirated.

4. Harvest of Murine Peyer's Patches and Isolation of Viable Yeast Colonies

1. At the appropriate time point post gavage, typically 4 hr, sacrifice mice using IACUC approved methods. Check for lack of responsiveness following a toe pinch, and use a secondary measure such as cervical dislocation to sacrifice the mouse. Additional time points may also be tested, as numerous studies have shown that efficiency and timing of uptake across the epithelium is particle dependent^38,39.
2. Lay the mouse with the abdomen fully exposed and sterilize the abdominal area by spraying with 70% EtOH. Make a transverse incision through the fur and skin with scissors, being careful not to damage any internal tissues. Manually pry the incision open further to expose the peritoneum, the thin serosal lining covering the abdominal organs. Gently lift the peritoneum and make a transverse incision to expose the intestines.
3. Carefully use blunt forceps to tease the small intestine away from the mesenteric arteries, fat, and other tissues. Expose the small intestine from the stomach, in the upper left quadrant of the mouse abdomen, to the cecum, the large pocket of intestinal tissue at the start of the large intestine.
4. Isolate the Peyer's patches by looking for 1-3 mm roughly circular patches of opaque tissue along the small intestine (Figure 7). Using curved dissection scissors, cut away the dome of the Peyer's patch, leaving margins to ensure that none of the surrounding lamina propria is collected.
   NOTE: Most mice have between 4-8 easily visible Peyer's patches. Performing the procedure in an area with direct overhead lighting will increase the ease with which Peyer's patches can be visualized.
5. Collect dissected Peyer's patches in complete Iscove's modified dulbecco's media (IMDM).
   NOTE: It is critical to use sterile technique and include antibiotics in the collection media in order to prevent gastrointestinal bacterial contamination of yeast plates.
6. Strain Peyer's patches through a 40 µm cell strainer to eliminate collection media. Wash Peyer's patches with fresh complete IMDM over a 50 ml tube and use a plunger from a 1 ml syringe to gently break up the Peyer's patches. Pellet strained cells by centrifugation at 1,800 rpm for 7 min. Aspirate supernatant and resuspend cells in a final volume of approximately 100 µl.
7. Apply strained cells onto selective yeast media and use a plate spreader to evenly distribute cells. Wrap plate edges in Parafilm and incubate plates upside down at 30 °C for 2 days to allow for growth of any viable yeast recovered from the murine Peyer's patches.
   NOTE: Further studies after recovery of yeast from Peyer' patches are necessary to confirm that the strains are able to deliver properly folded heterologous protein to these immune tissues. As described in 2.1.14, such methods may include immunoblotting, ELISA, or fluorescence microscopy to detect fluorescent proteins such as GFP^2,40.