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Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol
在粟酒裂殖酵母中采用醋酸锂转化方案进行标记基因的单步转换   

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Abstract

The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.

Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, Schizosaccharomyces pombe. In the following we describe how to swap the ura4+ marker to a kanMX6, natMX4, or hphMX4 marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the MX markers for nutritional markers, such as arg3+, his3+, leu1+ and ura4+.

Keywords: Schizosaccharomyces pombe(粟酒裂殖酵母), Selectable marker(可选标记基因), Marker switch(标记基因转换), Li-Acetate transformation(醋酸锂转化), Gene tagging(基因标签), Gene deletion(基因删除), Genetic manipulation(遗传操作)

Background

This single-step marker swap protocol for Schizosaccharomyces pombe allows for any tagged or mutated gene marked with an MX-type antibiotic marker to be swapped for a nutritional marker (cassettes containing the arg3+, his3+, leu1+, and ura4+ have been constructed) and to exchange genetic ura4+-markers for any MX-type antibiotic resistance marker (kanMX, natMX, and hphMX constructs have been tested for this study) (Lorenz et al., 2015a). Previously, this kind of approach was only feasible for MX-type antibiotic resistance markers (Sato et al., 2005; Hentges et al., 2005). Exchanging antibiotic resistance markers for each other already represented a basic set of useful genetic tools, the ura4+-to-MX as well as the arg3MX4, his3MX4, leu1MX4, and ura4MX4 marker swap cassettes expand this genetic toolbox for tagging and mutating genes in fission yeast (Lorenz et al., 2015a). The lithium acetate transformation protocol itself was described previously (Keeney and Boeke, 1994) and recently suggested modifications (http://listserver.ebi.ac.uk/pipermail/pombelist/2014/004012.html) were incorporated to provide a highly efficient procedure. Streamlining Schizosaccharomyces pombe strain construction in this way is time-saving and, therefore, will prove useful for fission yeast researchers.

Materials and Reagents

  1. Conical tubes:
    15 ml (Greiner Bio One, catalog number: 188261 )
    50 ml (Greiner Bio One, catalog number: 227261 )
  2. 1.5 ml centrifuge tubes (Greiner Bio One, catalog number: 616201 )
  3. 0.2 ml flat-cap PCR tubes (Corning, Axygen®, catalog number: PCR-02-C )
  4. Petri dishes (Greiner Bio One, catalog number: 633185 )
  5. BD Plastipak 10 ml syringes (BD, catalog number: 302188 )
  6. Millex-GP 33 mm diameter sterile syringe filter units (Polyethersulfone [PES] membrane, pore size: 0.22 µm) (EMD Millipore, catalog number: SLGPO33RS )
  7. Appropriate Schizosaccharomyces pombe strains: for a marker swap the strain must already carry a mutant or tagged gene marked with either an ura4+ or MX-type marker, such as kanMX, natMX, or hphMX (Bähler et al., 1998; Goldstein and McCusker, 1999). When introducing arg3MX4, his3MX4, leu1MX4, or ura4MX4 into a strain, this strain needs to be mutated for the respective gene at its original locus, e.g., arg3-D4 (Waddell and Jenkins, 1995), his3-D1 (Burke and Gould, 1994), leu1-32 (Keeney and Boeke, 1994), or ura4-D18 (Grimm et al., 1988).
  8. Plasmids: pALo120 (FYP2884), pALo121 (FYP2885), pALo122 (FYP2886), pFA6a-arg3MX4 (FYP2890), pFA6a-his3MX4 (FYP2891), pFA6a-leu1MX4 (FYP2892), and pFA6a-ura4MX4 (FYP2893) (Lorenz, 2015a and 2015b); plasmid can be obtained from the National BioResource Project (NRBP) of MEXT, Japan (please refer to FYP numbers when ordering).
  9. Sonicated salmon sperm DNA (Sigma-Aldrich, catalog number: 31149 )
  10. Agarose, molecular grade (Bioline, catalog number: BIO-41026 )
  11. Q5 high fidelity DNA polymerase (New England Biolabs, catalog number: M0491 )
  12. 5x Q5 reaction buffer (accessory part of Q5 high fidelity polymerase) (New England Biolabs, catalog number: M0491 )
  13. 10 mM dNTPs (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0191 )
  14. 10 µM oligonucleotides (to be used as primers in PCR reactions) (Sigma-Aldrich, USA)
  15. MilliQ H2O drawn from a MilliQ Advantage A10 system (EMD Millipore, catalog number: Z00Q0V0WW )
  16. CutSmart buffer (accessory part of restriction enzymes, can also be ordered separately under New England Biolabs, catalog number: B2704 )
  17. Restriction enzymes:
    XbaI restriction enzyme (New England Biolabs, catalog number: R0145 )
    BamHI-HF restriction enzyme (New England Biolabs, catalog number: R3136 )
    EcoRI-HF restriction enzyme (New England Biolabs, catalog number: R3101 )
    PvuII-HF restriction enzyme (New England Biolabs, catalog number: R3151 )
    SacI-HF restriction enzyme (New England Biolabs, catalog number: R3156 )
  18. Di-methyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  19. Ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10526383 )
  20. Sodium hydroxide (NaOH) pellets (anhydrous) (Sigma-Aldrich, catalog number: S8045 )
  21. Tris(hydroxymethyl)aminomethane (VWR, catalog number: 103156X )
  22. Glacial (100%) acetic acid (VWR, catalog number: 20104.334 )
  23. Yeast extract, microgranulated (ForMediumTM, catalog number: YEM02 )
  24. Glucose anhydrous (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10373242 )
  25. Adenine (Sigma-Aldrich, catalog number: A5665 )
  26. Uracil (ForMediumTM, catalog number: DOCO212 )
  27. Leucine (ForMediumTM, catalog number: DOCO156 )
  28. Lysine (Sigma-Aldrich, catalog number: L8662 )
  29. Histidine (Sigma-Aldrich, catalog number: H5659 )
  30. Arginine (Sigma-Aldrich, catalog number: A6969 )
  31. Agar granulated (ForMediumTM, catalog number: AGR10 )
  32. G418 disulfate (ForMediumTM, product number: G4181 )
  33. Nourseothricin-dihydrogen sulfate (clonNAT) (Werner BioAgents, catalog number: clonNAT)
  34. Hygromycin B (ForMediumTM, catalog number: HYG1000 )
  35. Yeast nitrogen base without amino acids and without ammonium sulfate (ForMediumTM, catalog number: CYN0502 )
  36. L-glutamic acid monosodium salt hydrate (sodium glutamate) (Sigma-Aldrich, catalog number: G5889 )
  37. 5 N hydrochloric acid (HCl) (VWR, catalog number: 30018.320 )
  38. Lithium acetate (Sigma-Aldrich, catalog number: L4158 )
  39. Polyethylene glycol (PEG) MW 3,350 (Sigma-Aldrich, catalog number: P4338 )
  40. DNA molecular weight marker, HyperLadderTM 1 kb (Bioline, catalog number: BIO-33025 )
  41. 0.5 M EDTA (pH 8.0) (see Recipes)
  42. 50x TAE gel electrophoresis buffer (see Recipes)
  43. YES (Yeast extract supplemented) broth (see Recipes)
  44. YES plates (see Recipes)
  45. Concentration of antibiotics in YES media (see Recipes)
  46. YNG (Yeast nitrogen base glutamate) plates (see Recipes)
  47. 1 M Tris/HCl (pH 7.5) (see Recipes)
  48. 1 M LiAc (pH 7.5) (see Recipes)
  49. 10x TE (pH 7.5) (see Recipes)
  50. LiAc/TE (see Recipes)
  51. 40% PEG (see Recipes)

Equipment

  1. Erlenmeyer glass flasks for culturing yeast (DURAN Group, catalog number: 2177144 )
  2. Infors HT multitron standard shaking incubator (Infors, model: Multitron Standard )
  3. Static incubator (Gallenkamp forced air incubator) (Weiss Technik UK)
  4. Water baths (Grant Instruments, catalog number: JBN5 )
  5. Eppendorf microcentrifuge (Eppendorf, model: 5424R )
  6. MJ Research PTC-100 programmable thermal cycler
  7. Nucleic acid gel electrophoresis system consisting of a gel electrophoresis chamber with a 7 x 10 cm tray (Bio-Rad Laboratories, model: Mini-Sub® Cell GT ) and a power supply (Bio-Rad Laboratories, model: PowerPacTM Basic Power Supply )
  8. NanoDrop 2000c UV/Vis-spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: ND-2000C )
  9. Sigma 4-16K centrifuge (Sigma Laborzentrifugen, model: Sigma 4-16K )
  10. Haemocytometer, type Neubauer improved (Marienfeld-Superior, catalog number: 0630010 )
  11. Leica DM500 microscope (Leica Microsystems, model: Leica DM500 )
  12. Classic Media 12 L autoclave (Prestige Medical, model: 210048 )

Procedure

  1. Generation of marker swap cassettes for transformation
    Marker swap cassettes to be used in the lithium acetate protocol below can be generated by two alternative means; by PCR amplification from or by restriction endonuclease digestion of the plasmids previously described in Lorenz (2015a and 2015b) (see below). Using appropriate modifications, this protocol can also be applied to constructs described elsewhere (Sato et al., 2005; Hentges et al., 2005; Gadaleta et al., 2013; Chen et al., 2015). After the PCR or restriction digest run 1/20 volume of each reaction on a 0.8% agarose gel in 1x TAE at 80 V for 45 min to verify that the reaction has worked properly (band sizes to be expected for each reaction are detailed below). The remainder of the PCR reaction or restriction is stopped by the addition of 2 μl of 0.5 M EDTA (pH 8.0). DNA concentration is measured on a NanoDrop 2000c UV/Vis-spectrophotometer, and an appropriate volume (maximum 20 μl) to result in 1-5 μg of cassette DNA is used for each transformation.
    1. ura4+ marker swap cassette amplification
      1. A 50 μl PCR reaction contains:
        100 ng of DNA from plasmid pALo120, pALo121, or pALo122 (Lorenz, 2015a and 2015b) to create cassettes by PCR for swapping an ura4+ marker to a kanMX6, natMX4, or hphMX4 marker, respectively.
        10 μl 5x Q5 reaction buffer
        200 μM dNTPs
        500 nM each of AL1forw (5’-agctacaaatcccactgg-3’) and AL1rev (5’-gtgatattgacgaaactttttg-3’) oligonucleotides as primers
        1 U Q5 high-fidelity DNA polymerase
        Make up to 50 μl by adding the appropriate amount of sterile MilliQ water (see Note 1).
      2. Use the following PCR programme:
        30 sec at 98 °C
        35 x (10 sec at 98 °C, 20 sec at 55 °C, 85 sec at 72 °C)
        120 sec at 72 °C
      3. Expected band sizes for transformation cassettes:
        pALo120 (ura4+-to-kanMX6 swapping cassette): 1.97 kb
        pALo121 (ura4+-to-natMX4 swapping cassette): 1.74 kb
        pALo122 (ura4+-to-hphMX4 swapping cassette): 2.2 kb
    2. Restriction digest to liberate ura4+ marker swap cassettes
      1. A 20 μl reaction contains:
        12 μg of DNA from plasmid pALo120, pALo121, or pALo122 (this will result in ~5 μg of marker swap cassette DNA) (Lorenz, 2015a and 2015b).
        2 µl CutSmart buffer
        2 µl XbaI restriction enzyme
        Make up to 20 μl by adding the appropriate amount of sterile MilliQ water (see Note 1).
      2. Incubate reaction at 37 °C for 1 h.
      3. Expected band sizes after restriction digest:
        pALo120: 1.98 kb (cassette) and 2.64 kb (vector backbone)
        pALo121: 1.75 kb (cassette) and 2.64 kb (vector backbone)
        pALo122: 2.2 kb (cassette) and 2.64 kb (vector backbone)
    3. MX marker swap cassette amplification
      1. A 50 μl PCR reaction contains:
        100 ng of DNA from plasmid pFA6a-arg3MX4, pFA6a-his3MX4, pFA6a-leu1MX4, or pFA6a-ura4MX4 (Lorenz, 2015a and 2015b) to create cassettes by PCR for swapping any MX-type marker to a arg3+, his3+, leu1+, or ura4+ marker, respectively.
        10 μl 5x Q5 reaction buffer
        200 μM dNTPs
        500 nM each of AL2forw (5’-gtttagcttgcctcgtccc-3’) and AL2rev (5’-gatggcggcgttagtatcg-3’) oligonucleotides as primers
        1 U Q5 high-fidelity DNA polymerase
        Make up to 50 μl by adding the appropriate amount of sterile MilliQ water (see Note 1).
      2. Use the following PCR programme:
        30 sec at 98 °C
        35 x (10 sec at 98 °C, 20 sec at 64 °C, 90 sec at 72 °C)
        120 sec at 72 °C
      3. Expected band sizes for transformation cassettes:
        pFA6a-arg3MX4: 2.54 kb
        pFA6a-his3MX4: 2.65 kb
        pFA6a-leu1MX4: 2.19 kb
        pFA6a-ura4MX4: 2.39 kb
    4. Restriction digest to liberate arg3MX4, leu1MX4, and ura4MX4 marker swap cassettes
      1. A 20 μl reaction contains:
        10 μg of DNA from plasmid pFA6a-arg3MX4, pFA6a-leu1MX4, or pFA6a-ura4MX4 (this will result in ~5 μg of marker swap cassette DNA) (Lorenz, 2015a and 2015b).
        2 µl CutSmart buffer
        1 µl BamHI-HF restriction enzyme
        1 µl EcoRI-HF restriction enzyme
        Make up to 20 μl by adding the appropriate amount of sterile MilliQ water (see Note 1).
      2. Incubate reaction at 37 °C for 1 h.
      3. Expected band sizes after restriction digest:
        pFA6a-arg3MX4: 2.6 kb (cassette) and 2.47 kb (vector backbone)
        pFA6a-leu1MX4: 2.24 kb (cassette) and 2.47 kb (vector backbone)
        pFA6a-ura4MX4: 2.44 kb (cassette) and 2.47 kb (vector backbone)
    5. Restriction digest to liberate the his3MX4 marker swap cassette
      1. A 20 μl reaction contains:
        10 μg of DNA from plasmid pFA6a-his3MX4 (this will result in ~5 μg of marker swap cassette DNA) (Lorenz, 2015a and 2015b).
        2 µl CutSmart buffer
        1 µl PvuII-HF restriction enzyme
        1 µl SacI-HF restriction enzyme
        Make up to 20 μl by adding the appropriate amount of sterile MilliQ water (see Note 1).
      2. Incubate reaction at 37 °C for 1 h.
      3. Expected band sizes after restriction digest:
        pFA6a-his3MX4: 2.72 kb (cassette) and 2.44 kb (vector backbone)

  2. Lithium acetate transformation
    1. Grow 100 ml of yeast cells from the strain(s) to be transformed in YES broth in a shaking incubator at 30 °C to a density of ~1 x 107 cells/ml (count with haemocytometer to establish density) (see Notes 2 and 3).
    2. Harvest by centrifugation (in two 50 ml conical tubes) at 1,900 x g for 3 min at 20 °C.
    3. Resuspend cells in 25 ml of sterile MilliQ water, and combine into one 50 ml conical tube.
    4. Harvest by centrifugation at 1,900 x g for 3 min at 20 °C.
    5. Resuspend cells in 5 ml LiAc/TE (see Recipes), centrifuge at 1,900 x g for 3 min at 20 °C.
    6. Resuspend cells in 1 ml LiAc/TE and transfer to 1.5 ml centrifuge tube.
    7. Centrifuge at 2,300 x g for 1 min at room temperature. Discard supernatant and resuspend cells at ~3 x 109 cells/ml in LiAc/TE (normally 300 µl, if starter culture was 100 ml of 1 x 107 cells/ml).
    8. To 100 µl of cells add 2 µl of sonicated salmon sperm DNA (10 mg/ml) and the DNA to transform (1-5 µg in a maximum volume of 20 µl). Mix well by carefully pipetting up and down.
    9. Incubate at room temperature for 10 min.
    10. Add 260 µl 40% PEG (see Recipes and Note 4) and incubate for 2-6 h at 30 °C. Mix well by carefully pipetting up and down.
    11. Add 43 µl DMSO (dimethyl sulfoxide), mix well by inverting the tube five times, and heat shock for 5 min at 42 °C (see Note 5).
    12. Plate suspension by evenly spreading onto 3 YES plates (~135 µl on each plate) without selection (see Recipes).
    13. After 24-48 h incubation at 30 °C, replica-plate these plates onto selective plates; either onto YES containing the corresponding antibiotic, or onto YNG lacking the corresponding supplement.
    14. Allow the selective plates to grow at 30 °C for several days until single colonies reach a diameter of 3-4 mm.
    15. Patch at least 20 colonies onto a fresh selective plate and incubate 24-48 h.
    16. Replica-plate onto a fresh selective plate and onto a plate selecting for the original marker, to ensure that the marker swap is correct (e.g., if the original strain was ura4+ and pALo120 was used to swap to kanMX6, the resulting strain must be resistant to G418 and unable to grow on media lacking uracil).

Data analysis

The critical step of the single-step marker swap procedure is to confirm that the markers have been truly exchanged in a transformant, i.e., that the new marker is correctly integrated at the target site, thereby removing the original marker. It is known that marker integration at its intended target site is not perfectly efficient in Schizosaccharomyces pombe, and a wide range of correct integration frequencies have been reported (Bähler et al., 1998; Sato et al., 2005). Correct integration is influenced by several parameters, including the chromatin status of the genomic site and length of sequence homologies flanking the marker cassettes. In the presented single-step marker swap cassettes flanking sequence homologies are between 200 and 400 bps, which is longer than the required minimum 80 bps (Bähler et al., 1998), but not long enough to enable 100% correct targeting in all cases. Swapping the ura4+ marker to any of the antibiotic MX markers occurred at frequencies between 22.9-100%. The correct integration efficiency of swapping an MX marker to a nutritional marker was tested at the meiotic gene hop1; this is more challenging due to the closed chromatin state at meiotic open reading frames (Bähler et al., 1998). Therefore, the frequency of exchanging a kanMX6-marker deleting hop1 with a nutritional marker was lower and varied between 15.3-36.6% (Lorenz, 2015a).

Notes

  1. When pipetting a PCR reaction or restriction digest, always start with the MilliQ water, add the buffer, then all other components, and finally the enzyme(s). Working from these protocols, it is important to calculate the amount of water required before one sets up the reaction.
  2. For the transformation protocol use yeast cells streaked onto the appropriate plate not longer than 7 days beforehand. Always check the genotype of yeast strains carefully before usage!
  3. It is crucial to grow the fission yeast cells to late logarithmic phase (~1 x 107 cells/ml) to achieve the most efficient transformation frequency. Growing cells to higher density will result in drastically reduced numbers of transformants, because cells enter stationary phase and develop a thicker cell wall (i.e., do not grow cells to higher than ~1 x 107 cells/ml and dilute).
  4. 40% PEG (see Recipes) needs to be prepared freshly on the day when the transformation is performed.
  5. The duration and temperature of the heat shock (step B11) is critical!

Recipes

  1. 0.5 M EDTA (pH 8.0) (1 L)
    186.1 g ethylenediaminetetraacetic acid (EDTA)
    Add 800 ml MilliQ water
    Adjust pH to 8.0 by adding NaOH pellets (solution will only become clear when pH > 7)
    Make up with MilliQ water to volume
    Sterilize by autoclaving
  2. 50x TAE gel electrophoresis buffer (1 L)
    242 g Tris(hydroxymethyl)aminomethane
    57.1 ml glacial acetic acid
    100 ml 0.5 M EDTA (pH 8.0)
    Make up with MilliQ water to volume
    Sterilize by autoclaving
    Use as 1x TAE by diluting 1:50 in MilliQ water
  3. YES (Yeast extract supplemented) broth (1 L)
    5 g yeast extract, microgranulated
    30 g glucose anhydrous
    250 mg adenine
    250 mg uracil
    250 mg leucine
    250 mg lysine
    250 mg histidine
    250 mg arginine
    Make up with MilliQ water to volume
    Sterilize by autoclaving
  4. YES plates (1 L, makes about 40 plates)
    As YES broth
    20 g agar granulated
    Sterilize by autoclaving
  5. Concentration of antibiotics in YES media (per 1 L)
    Note: Antibiotics are added to YES agar cooled down to 55 °C after autoclaving!
    200 mg G418 disulfate
    200 mg nourseothricin-dihydrogen sulfate (clonNAT)
    400 mg hygromycin B
  6. YNG (Yeast nitrogen base glutamate) plates (1 L, makes about 40 plates)
    1.9 g yeast nitrogen base (without amino acids and without ammonium sulfate)
    3.7 g L-glutamic acid monosodium salt hydrate (sodium glutamate)
    30 g glucose anhydrous
    20 g agar granulated
    75 mg adenine
    75 mg uracil
    75 mg leucine
    75 mg lysine
    75 mg histidine
    75 mg arginine
    Make up with MilliQ water to volume
    Sterilize by autoclaving
  7. 1 M Tris/HCl (pH 7.5) (1 L)
    121.1 g Tris(hydroxymethyl)aminomethane
    Add 800 ml MilliQ water
    Adjust pH to 7.5 by adding 5 N HCl
    Make up with MilliQ water to volume
    Sterilize by autoclaving
  8. 1 M LiAc (pH 7.5; 100 ml)
    20.4 g lithium acetate
    Add 70 ml MilliQ water
    Adjust pH to 7.5 with acetic acid, if necessary
    Make up with MilliQ water to volume
    Sterilize by autoclaving
  9. 10x TE (pH 7.5; 100 ml)
    2 ml 0.5 M EDTA (pH 8.0) (final concentration 0.01 M)
    10 ml 1 M Tris/HCl (pH 7.5) (final concentration 0.1 M)
    Make up with MilliQ water to volume
    Sterilize by autoclaving
  10. LiAc/TE (10 ml)
    1 ml 1 M LiAc (pH 7.5)
    1 ml 10x TE (pH 7.5) (0.1 M Tris, 0.01 M EDTA)
    8 ml sterile MilliQ water
  11. 40% PEG (10 ml)
    4 g PEG (polyethylene glycol) MW 3,350
    1 ml 1 M LiAc (pH 7.5)
    1 ml 10x TE (0.1 M Tris, 0.01 M EDTA)
    4 ml sterile MilliQ water
    Dissolve PEG completely (incubate at 37 °C for a few minutes, if necessary).
    Make up to 10 ml
    Filter sterilize

Acknowledgments

We acknowledge funding from the Biotechnology and Biological Sciences Research Council UK (BBSRC, Doctoral Training Grant BB/FO16964/1) and the College of Life Science and Medicine, University of Aberdeen, UK.

References

  1. Bähler, J., Wu, J. Q., Longtine, M. S., Shah, N. G., McKenzie, A., 3rd, Steever, A. B., Wach, A., Philippsen, P. and Pringle, J. R. (1998). Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14(10): 943-951.
  2. Burke, J. D. and Gould, K. L. (1994). Molecular cloning and characterization of the Schizosaccharomyces pombe his3 gene for use as a selectable marker. Mol Gen Genet 242(2): 169-176.
  3. Chen, Y., Chen, L., An, K., Wang, Y. and Jin, Q. (2015). A vector system for efficient and economical switching of a ura4+ module to three commonly used antibiotic marker cassettes in Schizosaccharomyces pombe. Yeast 32(11): 671-682.
  4. Gadaleta, M. C., Iwasaki, O., Noguchi, C., Noma, K. and Noguchi, E. (2013). New vectors for epitope tagging and gene disruption in Schizosaccharomyces pombe. Biotechniques 55(5): 257-263.
  5. Grimm, C., Kohli, J., Murray, J. and Maundrell, K. (1988). Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet 215(1): 81-86.
  6. Goldstein, A. L. and McCusker, J. H. (1999). Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15(14): 1541-1553.
  7. Hentges, P., Van Driessche, B., Tafforeau, L., Vandenhaute, J. and Carr, A. M. (2005). Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe. Yeast 22(13): 1013-1019.
  8. Keeney, J. B. and Boeke, J. D. (1994). Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136(3): 849-856.
  9. Lorenz, A. (2015a). New cassettes for single-step drug resistance and prototrophic marker switching in fission yeast. Yeast 32(12): 703-710.
  10. Lorenz, A. (2015b). Plasmid sequences: New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast. figshare, 10.6084/m9.figshare.1468419.
  11. Sato, M., Dhut, S. and Toda, T. (2005). New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe. Yeast 22(7): 583-591.
  12. Waddell, S. and Jenkins, J. R. (1995). arg3+, a new selection marker system for schizosaccharomyces pombe: application of ura4+ as a removable integration marker. Nucleic Acids Res 23(10): 1836-1837.

简介

利用不同的选择标记来标记或突变粟酒裂殖酵母中的多个基因的能力受到仅仅两种可选择标记的历史使用的阻碍,即 +和 kanMX6 ;后者赋予抗生素G418(遗传霉素)抗性。最近已经描述了更多的标记物,但是将它们引入酵母细胞通常需要从头开始施加应变。为了克服这个问题,我们和其他团队已经创建了具有与 + 和 kanMX6 的侧翼同源性的转换盒,这使得能够有效和省时的方式在现有的突变或标记的裂变酵母菌株中交换标记。
 在这里,我们提出了裂殖酵母,粟酒裂殖酵母(Schizosaccharomyces pombe)中醋酸锌转化的单步标记转换方案。以下我们将介绍如何将 ura4 + 标记交换到kanMX6 , natMX4 或 hphMX4标记,其分别对抗生素G418,海参(clonNAT)或潮霉素B提供抗性。我们还详细介绍了如何交换营养标记的任何 MX 标记,例如 arg3 + , his3 + , leu1 + 和 ura4 + 。

背景 这种用于粟酒裂殖酵母的单步骤标记交换方案允许将标记有类型的抗生素标记的任何标记或突变的基因替换为营养标记物(含有arg3 + , + ,并且已经构建了 ura4 + )并且为任何 MX交换遗传 ura4 + 已经测试了这种研究的型抗生素抗性标记( kanMX ,natMX 和 hphMX 结构)(Lorenz et al。,2015a)。以前,这种方法只适用于MX 型抗生素抗性标记(Sato et al。,2005; Hentges等人, 2005)。彼此交换抗生素抗性标记已经代表了一组基本的有用的遗传工具,即 ura4 + -to- MX arg3MX4 ,他的3MX4 , leu1MX4 和 ura4MX4 标记交换磁带扩展这个遗传工具箱,用于在裂变酵母中标记和突变基因(Lorenz等人,2015a)。以前描述了乙酸锂转化方案本身(Keeney和Boeke,1994),最近建议修改(http://listserver.ebi.ac.uk/pipermail/pombelist/2014/004012.html 被合并,以提供高效的程序。以这种方式简化粟酒裂殖酵母菌株施用是节省时间的,因此将证明对裂殖酵母研究人员有用。

关键字:粟酒裂殖酵母, 可选标记基因, 标记基因转换, 醋酸锂转化, 基因标签, 基因删除, 遗传操作

材料和试剂

  1. 圆锥管:
    15 ml(Greiner Bio One,目录号:188261)
    50ml(Greiner Bio One,目录号:227261)
  2. 1.5ml离心管(Greiner Bio One,目录号:616201)
  3. 0.2ml平头PCR管(Corning,Axygen,目录号:PCR-02-C)
  4. 培养皿(Greiner Bio One,目录号:633185)
  5. BD Plastipak 10 ml注射器(BD,目录号:302188)
  6. Millex-GP 33mm直径的无菌注射器过滤器单元(聚醚砜[PES]膜,孔径:0.22μm)(EMD Millipore,目录号:SLGPO33RS)
  7. 适当的粟酒裂殖酵母菌株:为了标记交换,菌株必须已经携带用 ura4 + 标记的突变体或标记基因MX 类型标记,例如 kanMX natMX hphMX (Bähler等人)。 ,1998; Goldstein和McCusker,1999)。当将 arg3MX4 ,他的3XX4 , leu1MX4 ura4MX4 引入应变时,需要为(Waddell和Jenkins,1995),他们的原始基因座(例如,,arg3-D4 ,his3-D1 (Burke and Gould, (Keeney和Boeke,1994),或者ura4-D18(Grimm等人,1988)。
  8. 质粒:pALo120(FYP2884),pALo121(FYP2885),pALo122(FYP2886),pFA6a-arg3MX4(FYP2890),pFA6a- his3MX4(FYP2891),pFA6a- leu1MX4(FYP2892)和pFA6a- ura4MX4(FY2893)(Lorenz,2015a和2015b);质粒可以从日本MEXT的国家生物资源项目(NRBP)获得(订购时请参考FYP号码)。
  9. 超声鲑鱼精子DNA(Sigma-Aldrich,目录号:31149)
  10. 琼脂糖,分子级(Bioline,目录号:BIO-41026)
  11. Q5高保真DNA聚合酶(New England Biolabs,目录号:M0491)
  12. 5x Q5反应缓冲液(Q5高保真聚合酶的附件)(New England Biolabs,目录号:M0491)
  13. 10mM dNTP(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0191)
  14. 10μM寡核苷酸(用作PCR反应中的引物)(Sigma-Aldrich,USA)
  15. 来自MilliQ Advantage A10系统(EMD Millipore,目录号:Z00Q0V0WW)的MilliQ H 2 O 2
  16. CutSmart缓冲液(限制酶的附加部分,也可以在New England Biolabs下分别订购,目录号:B2704)
  17. 限制酶:
    Xba I限制酶(New England Biolabs,目录号:R0145)
    HI-HF限制酶(New England Biolabs,目录号:R3136)
    RI-HF限制酶(New England Biolabs,目录号:R3101)
    II-HF限制酶(New England Biolabs,目录号:R3151)
    I-HF限制酶(New England Biolabs,目录号:R3156)
  18. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  19. 乙二胺四乙酸(EDTA)(Thermo Fisher Scientific,Fisher Scientific,目录号:10526383)
  20. 氢氧化钠(NaOH)颗粒(无水)(Sigma-Aldrich,目录号:S8045)
  21. 三羟甲基氨基甲烷(VWR,目录号:103156X)
  22. 冰醋酸(100%)乙酸(VWR,目录号:20104.334)
  23. 酵母提取物,微粒化(ForMedium TM ,目录号:YEM02)
  24. 无水葡萄糖(Thermo Fisher Scientific,Fisher Scientific,目录号:10373242)
  25. 腺嘌呤(Sigma-Aldrich,目录号:A5665)
  26. 尿嘧啶(ForMedium TM ,目录号:DOCO212)
  27. 亮氨酸(ForMedium TM ,目录号:DOCO156)
  28. 赖氨酸(Sigma-Aldrich,目录号:L8662)
  29. 组氨酸(Sigma-Aldrich,目录号:H5659)
  30. 精氨酸(Sigma-Aldrich,目录号:A6969)
  31. 琼脂颗粒(ForMedium TM ,目录号:AGR10)
  32. G418二硫酸盐(ForMedium ,商品号:G4181)
  33. 硝酸二氢硫酸(clonNAT)(Werner BioAgents,目录号:clonNAT)
  34. 潮霉素B(ForMedium TM,目录号:HYG1000)
  35. 无氨基酸和无硫酸铵的酵母氮碱(ForMedium TM,目录号:CYN0502)
  36. L-谷氨酸单钠盐水合物(谷氨酸钠)(Sigma-Aldrich,目录号:G5889)
  37. 5N盐酸(HCl)(VWR,目录号:30018.320)
  38. 乙酸锂(Sigma-Aldrich,目录号:L4158)
  39. 聚乙二醇(PEG)MW 3,350(Sigma-Aldrich,目录号:P4338)
  40. DNA分子量标记,HyperLadder 1 kb(Bioline,目录号:BIO-33025)
  41. 0.5 M EDTA(pH 8.0)(参见食谱)
  42. 50x TAE凝胶电泳缓冲液(见配方)
  43. YES(酵母提取物补充)肉汤(见食谱)
  44. YES板(见配方)
  45. 在YES媒体中浓缩抗生素(见食谱)
  46. YNG(酵母氮谷氨酸)板(见食谱)
  47. 1M Tris / HCl(pH 7.5)(参见食谱)
  48. 1 M LiAc(pH 7.5)(见配方)
  49. 10x TE(pH 7.5)(参见食谱)
  50. LiAc / TE(参见食谱)
  51. 40%PEG(参见食谱)

设备

  1. 用于培养酵母的Erlenmeyer玻璃烧瓶(DURAN Group,目录号:2177144)
  2. Infor HT多频标准振荡培养箱(Infors,型号:Multitron Standard)
  3. 静电培养箱(Gallenkamp强制空气培养箱)(Weiss Technik UK)
  4. 水浴(Grant Instruments,目录号:JBN5)
  5. Eppendorf微量离心机(Eppendorf,型号:5424R)
  6. MJ Research PTC-100可编程热循环仪
  7. 由具有7×10cm托盘的凝胶电泳室(Bio-Rad Laboratories,型号:Mini-Sub Cell Cell)组成的核酸凝胶电泳系统和电源(Bio-Rad Laboratories,型号:PowerPac TM 基本电源)
  8. NanoDrop 2000c UV / Vis-spectrophotometer(Thermo Fisher Scientific,Thermo Scientific ,型号:ND-2000C)
  9. Sigma 4-16K离心机(Sigma Laborzentrifugen,型号:Sigma 4-16K)
  10. 血细胞计数器,Neubauer型改进(Marienfeld-Superior,目录号:0630010)
  11. Leica DM500显微镜(Leica Microsystems,型号:Leica DM500)
  12. Classic Media 12升高压灭菌器(Prestige Medical,型号:210048)

程序

  1. 生成用于转换的标记交换盒
    用于以下乙酸锂方案的标记交换盒可以通过两种替代方法产生;通过以前在Lorenz(2015a和2015b)中描述的质粒的限制性内切核酸酶消化或通过PCR扩增(见下文)进行PCR扩增。使用适当的修改,该协议也可以应用于其他地方描述的构造(Sato et al。,2005; Hentges等人,2005; Gadaleta等人, em,2013; Chen等人,2015)。在PCR或限制性消化后,在80V的1×TAE的0.8%琼脂糖凝胶上进行1/20体积的每个反应45分钟以验证反应是否正常工作(每个反应的预期带尺寸详述如下) 。通过加入2μl0.5M EDTA(pH8.0)来终止PCR反应或限制性的其余部分。在NanoDrop 2000c UV / Vis分光光度计上测量DNA浓度,每次转化使用适当的体积(最大20μl)以产生1-5μg盒式DNA。
    1. ura4 + 标记交换盒放大
      1. 50μlPCR反应包含:
        来自质粒pALo120,pALo121或pALo122(Lorenz,2015a和2015b)的100ng DNA通过PCR产生用于将 ura4 + 标记置换到kanMX6的盒, natMX4 hphMX4 标记。
        10μl5x Q5反应缓冲液
        200μMdNTPs
        作为引物的AL1forw(5'-agctacaaatcccactgg-3')和AL1rev(5'-gtgatattgacgaaactttttg-3')寡核苷酸各500nM, 1 U Q5高保真DNA聚合酶
        通过加入适量的无菌MilliQ水补足50μl(见注1)
      2. 使用以下PCR程序:
        98°C 30秒
        35×(98℃10秒,55℃20秒,72℃85秒)
        72°C 120秒,
      3. 转换盒的期望带尺寸:
        pALo120( ura4 + -to- kanMX6 交换盒):1.97 kb
        pALo121( ura4 + -to- natMX4 交换磁带):1.74 kb
        pALo122( ura4 + -to- hphMX4 交换盒):2.2kb
    2. 限制性消化释放 ura4 + 标记交换磁带
      1. 20μl反应物含有:
        12μg来自质粒pALo120,pALo121或pALo122的DNA(这将导致约5μg的标记交换盒DNA)(Lorenz,2015a和2015b)。
        2μlCutSmart缓冲液
        2μlXba I限制酶
        加入适量的无菌MilliQ水补足20μl(见注1)
      2. 在37℃下孵育反应1小时
      3. 限制性消化后预期的胶带尺寸:
        pALo120:1.98kb(盒)和2.64kb(载体骨架)
        pALo121:1.75kb(盒)和2.64kb(载体骨架)
        pALo122:2.2kb(盒)和2.64kb(载体骨架)
    3. MX 标记交换磁带放大器
      1. 50μlPCR反应包含:
        来自质粒pFA6a-arg3MX4的100ng DNA,pFA6a- his3MX4,pFA6a- leu1MX4或pFA6a-ura4MX4 (Lorenz,2015a和2015b)通过PCR创建盒子,将任何MX型标记交换到arg3 + his3 + leu1 + ura4 + 标记。
        10μl5x Q5反应缓冲液
        200μMdNTPs
        作为引物的AL2forw(5'-gtttagcttgcctcgtccc-3')和AL2rev(5'-gatggcggcgttagtatcg-3')寡核苷酸各500nM 1 U Q5高保真DNA聚合酶
        通过加入适量的无菌MilliQ水补足50μl(见注1)
      2. 使用以下PCR程序:
        98°C 30秒
        35×(98℃10秒,64℃20秒,72℃90秒)
        72°C 120秒,
      3. 转换盒的期望带尺寸:
        pFA6a-> arg3MX4 :2.54 kb
        pFA6a-他的3MX4 :2.65kb
        pFA6a- leu1MX4 :2.19 kb
        pFA6a- ura4MX4 :2.39kb
    4. 限制性摘要解放arg3MX4 , leu1MX4 ura4MX4 标记交换磁带
      1. 20μl反应物含有:
        来自质粒pFA6a-arg3MX4,pFA6a- leu1MX4或pFA6a- ura4MX4的10μgDNA(这将导致〜5μg的标记交换盒式DNA)(Lorenz,2015a和2015b)。
        2μlCutSmart缓冲液
        1μlBamHI HI-HF限制酶
        1μlEco-RI-HF限制酶
        加入适量的无菌MilliQ水补足20μl(见注1)
      2. 在37℃下孵育反应1小时
      3. 限制性消化后预期的胶带尺寸:
        pFA6a-> arg3MX4 :2.6kb(盒式)和2.47kb(载体骨架)
        pFA6a- leu1MX4:2.24kb(盒式)和2.47kb(载体骨架)
        pFA6a- ura4MX4:2.44kb(盒)和2.47kb(载体骨架)
    5. 限制性消化以释放他/她的3XX4标志交换盒
      1. 20μl反应物含有:
        来自质粒pFA6a- his3MX4的10μgDNA(这将导致约5μg的标记交换盒DNA)(Lorenz,2015a和2015b)。
        2μlCutSmart缓冲液
        1μlPvu II-HF限制酶
        1μlSac I / HF限制酶
        加入适量的无菌MilliQ水补足20μl(见注1)
      2. 在37℃下孵育反应1小时
      3. 限制性消化后预期的胶带尺寸:
        pFA6a- his3MX4 :2.72kb(盒式)和2.44kb(载体骨架)

  2. 乙酸锂转化
    1. 在振荡培养箱中在30℃下将来自待转化菌株的100ml酵母细胞培养至约1×10 7个细胞/ ml的密度(用血细胞计数器计数至建立密度)(见注2和3)。
    2. 通过在20℃下以1,900×g离心(在两个50ml锥形管中)收获3分钟。
    3. 将细胞重悬于25毫升无菌MilliQ水中,并结合成一个50ml锥形管
    4. 在20℃下以1,900×g离心收获3分钟。
    5. 将细胞重悬在5ml LiAc / TE(参见食谱)中,在20℃下以1,900×g离心3分钟。
    6. 将细胞重悬于1ml LiAc / TE中并转移到1.5ml离心管中
    7. 在室温下以2,300×g离心1分钟。弃去上清并在LiAc / TE中以〜3×10 9个细胞/ ml重悬细胞(通常300μl,如果起始培养物为100ml 1×10 7个细胞/ ml)
    8. 向100μl细胞中加入2μl超声处理的鲑鱼精DNA(10毫克/毫升)和DNA转化(1-5微克,最大体积为20微升)。通过仔细地上下移动来混合。
    9. 在室温下孵育10分钟
    10. 加入260μl40%PEG(参见食谱和注释4),并在30℃下孵育2-6小时。通过仔细地上下移动来混合。
    11. 加入43μlDMSO(二甲基亚砜),将管倒置5次,并在42℃下热休克5分钟(参见附注5)。
    12. 将板悬浮液均匀分布在3个YES板上(每个板上约135μl),无需选择(参见食谱)。
    13. 在30℃孵育24-48小时后,将这些板复制到选择性板上;要么是含有相应抗生素的YES,要么对YNG缺乏相应的补充。
    14. 允许选择性板在30℃下生长数天,直到单个菌落达到3-4mm的直径。
    15. 将至少20个菌落补充到新鲜的选择性板上并孵育24-48小时。
    16. 将复制板置于新鲜的选择性板上并选择原始标记的板上,以确保标记交换正确(例如,如果原始菌株为 ura4 + 和pALo120用于交换到kanMX6,所得菌株必须耐G418抗性,并且在缺乏尿嘧啶的培养基上无法生长。

数据分析

单步标记交换程序的关键步骤是确认标记已经在转化体(即)中真正交换,新的标记正确集成在目标位点,从而去除原始标记已知在其预期目标位点处的标记整合在粟酒裂殖酵母中不是完全有效的,并且已经报道了广泛的正确的积分频率(Bähler等人, 1998; Sato等人,2005)。正确的整合受几个参数的影响,包括基因组位点的染色质状态和标记盒侧翼序列同源性的长度。在所提出的单步标记交换盒中,侧翼序列同源性在200和400bps之间,其长于所需的最小80bps(Bähler等人,1998),但不足以使得在所有情况下100%正确的定位。将 ura4 + 标记交换到任何抗生素MX 标记以22.9-100%之间的频率发生。在减数分裂基因hop1上测试将MX标记物交换到营养标记物的正确的整合效率;由于减数分裂开放阅读框处的闭合染色质状态,这是更具挑战性的(Bähler等人,1998)。因此,与营养标记物交换kanMX6 -marker删除hop1 的频率较低,在15.3-36.6%之间变化(Lorenz,2015a)。

笔记

  1. 当移取PCR反应或限制性消化时,始终从MilliQ水开始,加入缓冲液,然后加入所有其他组分,最后加入酶。从这些协议工作,重要的是在设置反应之前计算所需的水量。
  2. 对于转化方案,使用预先不超过7天条纹到合适平板的酵母细胞。在使用之前,请仔细检查酵母菌株的基因型!
  3. 将裂殖酵母细胞生长至晚期对数期(〜1×10 7个细胞/ ml)是至关重要的,以实现最有效的转化频率。由于细胞进入固定相并形成较厚的细胞壁(,不生长细胞至高于1×10 ),细胞生长至较高密度将导致转化体数量急剧减少。 7细胞/ ml,稀释)。
  4. 在进行转化的当天,需要新鲜地制备40%PEG(参见食谱)。
  5. 持续时间和温度 的热休克(步骤B11)至关重要!

食谱

  1. 0.5M EDTA(pH8.0)(1L)
    186.1g乙二胺四乙酸(EDTA)
    加入800毫升MilliQ水
    通过加入NaOH颗粒调节pH至8.0(当pH> 7时溶液将变得清晰)
    弥补MilliQ水量
    高压消毒灭菌
  2. 50倍TAE凝胶电泳缓冲液(1升)
    242g Tris(羟甲基)氨基甲烷
    57.1毫升冰醋酸
    100ml 0.5M EDTA(pH8.0)
    弥补MilliQ水量
    高压消毒灭菌
    在MilliQ水中稀释1:50,作为1x TAE使用
  3. YES(酵母提取物补充)肉汤(1L)
    5克酵母提取物,微粒化的
    30克无水葡萄糖 250毫克腺嘌呤
    250毫克尿嘧啶
    250毫克亮氨酸
    250毫克赖氨酸
    250毫克组氨酸
    250毫克精氨酸
    弥补MilliQ水量
    高压消毒灭菌
  4. YES板(1升,约40块板)
    作为YES肉汤
    20克琼脂造粒
    高压消毒灭菌
  5. YES媒体中浓度的抗生素(每1升)
    注意:高压灭菌后,将抗生素加入到冷却至55℃的YES琼脂中。

    200mg G418二硫酸盐
    200毫克海洛替清 - 硫酸二氢盐(clonNAT)
    400毫克潮霉素B
  6. YNG(酵母氮谷氨酸)板(1L,制成约40个板)
    1.9克酵母氮碱(不含氨基酸,不含硫酸铵)
    3.7克L-谷氨酸单钠盐水合物(谷氨酸钠)
    30克无水葡萄糖 20克琼脂造粒
    75毫克腺嘌呤
    75毫克尿嘧啶
    75毫克亮氨酸
    75毫克赖氨酸
    75毫克组氨酸
    75毫克精氨酸
    弥补MilliQ水量
    高压消毒灭菌
  7. 1M Tris / HCl(pH7.5)(1L)
    121.1g三(羟甲基)氨基甲烷
    加入800毫升MilliQ水
    通过加入5N HCl将pH调节至7.5 弥补MilliQ水量
    高压消毒灭菌
  8. 1M LiAc(pH7.5; 100ml)
    20.4克醋酸锂
    加入70毫升MilliQ水
    如果需要,用乙酸调节pH至7.5 弥补MilliQ水量
    高压消毒灭菌
  9. 10x TE(pH 7.5; 100ml)
    2ml 0.5M EDTA(pH8.0)(终浓度0.01M) 10ml 1M Tris / HCl(pH7.5)(终浓度0.1M)
    弥补MilliQ水量
    通过高压灭菌消毒
  10. LiAc / TE(10ml)
    1 ml 1M LiAc(pH 7.5)
    1 ml 10x TE(pH 7.5)(0.1M Tris,0.01M EDTA) 8毫升无菌MilliQ水
  11. 40%PEG(10ml)
    4g PEG(聚乙二醇)MW 3,350
    1 ml 1M LiAc(pH 7.5)
    1ml 10x TE(0.1M Tris,0.01M EDTA) 4毫升无菌MilliQ水
    完全溶解PEG(如有必要,在37℃下孵育数分钟)。
    补充10 ml
    过滤灭菌

致谢

我们承认英国生物技术和生物科学研究理事会(BBSRC,博士培训授权BB / FO16964 / 1)和英国阿伯丁大学生命科学与医学学院的资助。

参考文献

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  2. Burke,JD and Gould,KL(1994)。  分子克隆和表征粟酒裂殖酵母基因,用作可选择的标记。分子Gen Genet 242(2):169-176。
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  5. Grimm,C.,Kohli,J.,Murray,J.and Maundrell,K。(1988)。粟酒裂殖酵母的遗传工程:使用 ura4 基因作为选择标记的基因破坏和置换系统。 Mol Gen Genet 215(1):81-86。
  6. Goldstein,AL和McCusker,JH(1999)。  三酵母菌(Saccharomyces cerevisiae)基因破坏的新的主要药物耐药性盒。 15(14):1541-1553。
  7. Hentges,P.,Van Driessche,B.,Tafforeau,L.,Vandenhaute,J.and Carr,AM(2005)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/16200533"target ="_ blank">三种新型抗生素标记盒,用于在粟酒裂殖酵母中的基因破坏和标记转换。 酵母 22(13):1013-1019。
  8. Keeney,JB和Boeke,JD(1994)。 Efficient粟酒裂殖酵母中的 leu1-32 和 ura4-294 的目标整合。遗传学 136(3 ):849-856。
  9. Lorenz,A.(2015a)。新单盒 - 裂殖酵母中的耐药性和原养分标记转换。 酵母 32(12):703-710。
  10. Lorenz,A.(2015b)。质粒序列:分裂酵母中的耐药性和原养分生物标记切换。,无花果,10.6084 / m9.figshare.1468419。
  11. Sato,M.,Dhut,S.and Toda,T。(2005)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15942936"目标="_ blank">用于基因破坏和粟酒裂殖酵母中的表位标签的新的耐药盒 22(7):583-591。 br />
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引用:Brown, S. D. and Lorenz, A. (2016). Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol. Bio-protocol 6(24): e2075. DOI: 10.21769/BioProtoc.2075.
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