Multiple Modification of Chromosomal Loci Using URA5.3 Selection Marker in the Unicellular Red Alga Cyanidioschyzon merolae
在单细胞红藻 Cyanidioschyzon merolae中利用URA5.3选择标记对染色体基因进行多重修饰   

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Plant Cell Physiology
Nov 2018

 

Abstract

The unicellular red alga Cyanidioschyzon merolae has been used as a eukaryotic photosynthetic model for various basic and applied studies. Although the nuclear genome of C. merolae can be modified by homologous recombination with exogenously introduced DNA, it has been difficult to modify multiple chromosome loci within the same strain because of the limited number of available positive selection markers. Recently, we reported a modified URA5.3 gene cassette (URA5.3T), which can be used repeatedly for nuclear genome transformation using the pMKT plasmid vectors for epitope tagging (3x FLAG- or 3x Myc-) of nuclear-encoded proteins. In addition, these plasmid vectors can also be used to knock out multiple genes one by one. This report describes the construction of DNA fragments for transformation and the detailed transformation procedure.

Keywords: Cyanidioschyzon merolae (Cyanidioschyzon merolae), Epitope tagging (表位标记), Gene knocking out (基因敲除), Homologous recombination (同源重组), Selectable marker recycling (可筛选标记重复利用), URA5.3 (URA5.3)

Background

The standard method of transformation in C. merolae uses the uracil-auxotrophic strains M4 (Minoda et al., 2004) or T1 (Taki et al., 2015), which harbor a frameshift or a complete deletion mutation in the gene URA5.3 (CMK046C). The gene URA5.3 encodes a protein composed of an orotate phosphor ribosyl transferase (OPRTase) domain and an orotidine-5'-phosphate decarboxylase (OMPdecase) domain, which synthesizes uridine-5'-monophosphate from orotate. The transformant is selected by introducing URA5.3 as a selection marker into the uracil-auxotrophic strain. A recent study has reported the chloramphenicol resistance CAT gene as a second selectable marker gene for the nuclear transformation experiment in C. merolae (Fujiwara et al., 2017). However, it is difficult to modify more than three genome loci. Thus, we developed the URA5.3 selection marker gene recycling system using a modified URA5.3 gene, named URA5.3T (Takemura et al., 2018). The URA5.3T includes the URA5.3 terminator sequence (462 bp) at the 5’ end of the URA5.3 promoter-terminator cassette to make homologous repeats, the epitope tag region which encodes the recognition sequence for three different proteases (TEV, Factor Xa and thrombin), and a 3x FLAG or 3x Myc tag-encoding sequence (Figure 1). The URA5.3 promoter-terminator cassette is eliminated by frequently occurring DNA excision through the homologous repeat sequences located on both sides of URA5.3T. In this method, the URA5.3 eliminated transformant is selected using 5-fluoroorotic acid (5-FOA), which is converted to cytotoxic 5-fluorouracil via URA5.3 activity. The marker-eliminated transformant can be used for the next round transformation experiment using the URA5.3T marker gene (Figure 1). In this report, we present the procedure for construction of the transformation template DNA, which includes the recycling marker URA5.3T, for the purpose of gene tagging or gene knock out.


Figure 1. Outline from transformation to URA5.3 marker elimination using URA5.3T. The DNA includes the homologous regions for the genome-specific locus (grey box) and the URA5.3T. The URA5.3T includes the epitope tag region, a homologous repeat sequence (red hatched box) and the URA5.3 promoter-terminator cassette. The epitope tag region contains the recognition sequence for three different proteases (TEV, Factor Xa and thrombin) and a 3x FLAG or 3x Myc tag-encoding sequence.

Materials and Reagents

  1. Pipette tips (NIPPON Genetics Co., Ltd., catalog numbers: FG-102, FG-301, FG-401)
  2. Toothpicks
  3. Cuvettes (As One Corporation., catalog number: 1-2855-02)
  4. 1.5 ml Microtube (BM Equipment Co., Ltd., catalog number: NT-175)
  5. 50 ml Centrifuge tube (Corning Incorporated, catalog number: 352196)
  6. 24-well tissue culture test plate (BM Equipment Co., Ltd., catalog number: 92424)
  7. Sterilized plastic plate (Φ 90 mm) (As One Corporation, catalog number: GD90-15)
  8. AnaeroPouch (Mitsubishi Gas, catalog number: 2-3764-02)
  9. AnaeroPack (Mitsubishi Gas, catalog number: 2-3765-01)
  10. C. merolae T1 strain (Taki et al., 2015)
  11. Escherichia coli DH5α (Takara Bio, catalog number: 9057)
  12. Wild type C. merolae DNA
  13. Salmon sperm DNA (FUJIFILM Wako Pure Chemical Industries Corporation, catalog number: 047-17322)
  14. Plasmid vector pMKT (Takemura et al., 2018; available upon request to authors)
  15. KOD-Plus-Neo DNA polymerase (Toyobo, catalog number: KOD-401)
  16. GoTaq DNA polymerase (Promega, catalog number: M7123)
  17. StuI (NEB, catalog number: R0187S)
  18. DpnI (Takara Bio, catalog number: 1235A)
  19. Specific primers [set for amplifying the Upstream and Downstream fragments with the homologous sequence for the StuI-digested pMKT (see Figure 2A), ordered at Integrated DNA Technologies (IDT) Inc.]
  20. Confirmation primer sets (annealing to the outside sequence of the homologous regions, see Figure 3, ordered at Integrated DNA Technologies (IDT) Inc.)
  21. Primer sets for colony PCR (ordered at Integrated DNA Technologies (IDT) Inc.):
    Forward primer for up: 5’-CGAGGTCGACGGTATCGATAAGC-3’
    Reverse primer for up: 5’-GATCTAGTAACATAGATGACACCGC-3’
    Forward primer for down: 5’-GCTGCTAGGGATTGTGGCGCGA-3’
    Reverse primer for down: 5’-CCGCTCTAGAACTAGTGGATCCC-3'
  22. In-fusion HD cloning kit (Takara Bio, catalog number: 639648)
  23. Phenol, Crystals (FUJIFILM Wako Pure Chemical Corporation, catalog number: 169-22445)
  24. Chloroform (FUJIFILM Wako Pure Chemical Corporation, catalog number: 035-02616)
  25. Isoamyl alcohol (3-Methyl-1-butanol) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 135-12015)
  26. Trizma® base Primary Standard and Buffer, crystalline (Sigma-Aldrich, catalog number: T1503-500G)
  27. HCl (FUJIFILM Wako Pure Chemical Corporation, catalog number: 081-03475)
  28. Sodium dodecyl sulfate (SDS) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 194-13985)
  29. NaOH (FUJIFILM Wako Pure Chemical Corporation, catalog number:199-4895)
  30. 8-quinolinol (FUJIFILM Wako Pure Chemical Corporation, catalog number: 178-00752)
  31. Agarose ME (Iwai Chemical Company, catalog number: 50013R)
  32. Wizard SV gel and PCR clean-up system (Promega, catalog number: A9281)
  33. Polyethylene glycol 4000 (FUJIFILM Wako Pure Chemical Corporation, catalog number: 5322-68-3)
  34. Cornstarch (Kawamitsu-Bussan, catalog number: 4901 486 02701 6)
  35. Top starch solution (Takemura et al., 2019)
  36. Gellan gum (FUJIFILM Wako Pure Chemical Corporation, catalog number: 073-03071)
  37. Uracil (Sigma-Aldrich, catalog number: U0750-5G)
  38. 5-Fluoroorotic acid monohydrate (5-FOA) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 066-03663)
  39. Sodium acetate (FUJIFILM Wako Pure Chemical Corporation, catalog number: 190-01071)
  40. Ethanol (FUJIFILM Wako Pure Chemical Corporation, catalog number: 055-00457)
  41. (NH4)2SO4 (FUJIFILM Wako Pure Chemical Corporation, catalog number: 019-03435)
  42. MgSO4∙7H2O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 138-00415)
  43. H2SO4 (FUJIFILM Wako Pure Chemical Corporation, catalog number: 192-04696)
  44. H3BO3 (FUJIFILM Wako Pure Chemical Corporation, catalog number: 021-02195)
  45. MnCl2∙4H2O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 133-00725)
  46. ZnCl2 (FUJIFILM Wako Pure Chemical Corporation, catalog number: 268-01022)
  47. Na2MoO4∙2H2O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 198-02471)
  48. CoCl2∙6H2O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 038-03681)
  49. CuCl2∙2H2O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 039-04135)
  50. KH2PO4 (FUJIFILM Wako Pure Chemical Corporation, catalog number: 169-04245)
  51. CaCl2∙2H2O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 038-19735)
  52. FeCl3∙6H2O (FUJIFILM Wako Pure Chemical Corporation, catalog number: 090-02802)
  53. Na2EDTA (FUJIFILM Wako Pure Chemical Corporation, catalog number: 345-01865)
  54. DNA solution (see Recipes)
  55. PEG solution (see Recipes)
  56. MA2 medium (Kobayashi et al., 2010, see Recipes)
    1. MA2 solution I
    2. A6 minor salts
    3. MA2 solution II
    4. MA2 solution III
    5. MA2 solution IV
  57. MA2 solid gellan gum plate (see Recipes)
  58. DNA extraction buffer (see Recipes)
  59. Neutralized phenol (see Recipes)

Equipment

  1. Pipettes (Gilson, Inc., catalog numbers: F123600, F123601, F123602)
  2. Refrigerated centrifuge (Koki Holdings Co., Ltd., model: CF16RXII)
  3. Microcentrifuge (TOMY Seiko Co., Ltd., model: MX150)
  4. 500 ml flask (IWAI, catalog number: 4442FK500)
  5. Heat Block (Chiyoda Science Co., Ltd., model: MiniT-100)
  6. Bio incubator (TOMY Seiko Co., Ltd., model: CLE-303)
  7. NanoDrop (Thermo Fisher Scientific Inc., model: ND-LITE)
  8. Thermal Cycler (Bio-Rad Laboratories, Inc., model: T100 Thermal Cycler)
  9. Electrophoresis apparatus (Atto Corporation, model: WSE-1710)
  10. FAS gel imager (Nippon Genetics Co., Ltd., model: FAS-IV)
  11. Spectrophotometer (Beckman coulter, Inc., model: DU730, A23616)

Procedure

  1. Construction of the introducing DNA fragment
    Overview of the construction: PCR-amplified DNA fragments (Upstream and Downstream of homologous regions for the genome-specific locus) and StuI-digested pMKT (including two fragments Digested vector L and S) are connected using the In-fusion system to generate the pMKT-UPandDOWN. The resultant plasmid is used as the template DNA for amplification of the introducing DNA fragment by PCR (Figure 2).
    PCR amplification of DNA fragments (Figure 2A):
    1. Specific primer sets are designed as given below to amplify the DNA fragments (500-1,500 bp each) for homologous recombination to the genome-specific locus. Upstream and Downstream DNA fragments do not need to share the same length. For tagging, the upstream DNA fragment should include an open reading frame devoid of the termination codon, which is fused in frame to the epitope tag. Each colored region (20 bp) is the homologous sequence of the end of relevant StuI-digested pMKT (The colors correspond to Figure 2A) and each repeated ‘Ns’ (20-25 bp) indicates the annealing sequence to amplify the Upstream and Downstream DNA fragments. The Tm of the annealing sequence (‘Ns’) of each primer should be designed to be above 63 °C.
      Upstream Forward (UF):
      5’-GACACGTGAATTTAAATAGGNNNNNNNNNNNNNNNNNNNN-3’
      Upstream Reverse (UR):
      5’-TGAAAATAAAGATTTTCAGGNNNNNNNNNNNNNNNNNNNN-3’
      Downstream Forward (DF):
      5’-AGGGATTGTGGCGCGACAGGNNNNNNNNNNNNNNNNNNNN-3’
      Downstream Reverse (DR):
      5’-GACACGTGAATTTAAATAGGNNNNNNNNNNNNNNNNNNNN-3’
    2. PCR reactions are carried out using KOD-Plus-Neo DNA polymerase under the following reaction conditions: 94 °C for 2 min, followed by 30 cycles of 10 s at 98 °C, 30 s/1 kb at 68 °C, and a final extension of 2 min/1 kb at 68 °C with specific primers sets (UF/UR for Upstream and DF/DR for Downstream) and 50 ng wild type C. merolae DNA as a template. PCR-amplified DNA fragments are purified using the Wizard SV Gel and PCR Clean-Up System following the manufacturer’s protocol, and the concentration of DNA is measured.
      Note: We routinely obtain reproducible amplification results by the above condition. The PCR results should be checked by gel electrophoresis to confirm the amplification.

  2. pMKT vector digestion by StuI to obtain two DNA fragments, here named Digested vector L (Long, approximately 3.6 kbp) and S (Short, approximately 3.0 kbp, Figure 2B)
    1. Incubate 1 μg of pMKT DNA in 50 μl 1x CutSmart Buffer (i.e., provided buffer of StuI) containing 10 units of StuI at 37 °C for 1 h.
    2. Add 100 μl of dH2O and 150 μl of Phenol:Chloroform:Isoamyl alcohol = 25:24:1 solution and mix vigorously. Centrifuge at 12,000 x g for 10 min at room temperature. Take 150 μl of supernatant and add 15 μl of 3 M sodium acetate and 375 μl of 100% EtOH. Mix vigorously and centrifuge at 12,000 x g for 30 min at 4 °C. Discard the supernatant, add 75% EtOH to the precipitate, and centrifuge at 12,000 x g for 5 min at 4 °C. Discard the supernatant, completely remove the remaining drops by pipette after flush centrifugation, and air dry the pellet briefly on the bench at room temperature. Then add 20 μl of dH2O and dissolve the precipitated DNA. Measure the DNA concentration.
      Note: When using phenol-chloroform-isoamyl alcohol, personal protective equipment needs to be used according to local health and safety regulations. Always work under a fume hood.

  3. In-fusion reaction to generate the template plasmid (Figure 2C)
    1. Gently mix 5 μl of DNA mixture [StuI-digested pMKT (Digested vector L and S):upstream:downstream ≈ 100 fmol:50 fmol:50 fmol] and 5 μl of 2x In-fusion reaction mix. Incubate the reaction mixture at 50 °C for 15 min and then cool it down on ice.
    2. Transform Escherichia coli DH5α with the reaction mixture by using a chemical transformation method as common use. Find the expected plasmid construct by colony PCR. PCR reactions are carried out using GoTaq DNA polymerase under the following reaction conditions: 95 °C for 3 min, followed by 30 cycles of 30 s at 95 °C, 30 s at 55 °C, 1 min/1 kb at 72 °C, and a final extension of 2 min at 72 °C with primers sets (Forward primer for up: 5’-CGAGGTCGACGGTATCGATAAGC-3’/Reverse primer for up: 5’-GATCTAGTAACATAGATGACACCGC-3’ for the upstream sequence, and Forward primer for down: 5’-GCTGCTAGGGATTGTGGCGCGA-3’/Reverse primer for down: 5’-CCGCTCTAGAACTAGTGGATCCC-3’ for the downstream sequence). Subsequently, the positive candidates are examined by DNA sequence analysis with primer sets for colony PCR. 
    3. Extract and purify the plasmid DNA (pMKT-UPandDOWN).

  4. Amplification of DNA for transformation by PCR to prepare the linearized DNA fragment and completely remove the template circular plasmid DNA (Figure 2D)
    Note: Inclusion of the template plasmid DNA frequently may result in random integration of the plasmid into the genome. Thus, the template plasmid DNA should be carefully removed.


    Figure 2. Overview of the template vector (pMKT-UPandDOWN) and DNA constructs for transformation. A. DNA regions (Upstream and Downstream) corresponding to the target gene are amplified by PCR with the specific primer sets having the 20 bp homologous sequence for the pMKT (shown as red, blue, green and yellow). B. Digestion of the pMKT vector with StuI enzyme generates 2 DNA fragments named Digested vectors L and S after their lengths (L-long; S-short). The red hatched areas indicate the homologous repeat sequence (i.e., URA5.3 terminator region). C. Four DNA fragments (Digested vector L, Digested vector S, Upstream and Downstream) are connected in one reaction tube using the In-fusion system, to make a plasmid pMKT-UPandDOWN). D. The DNA fragment for transformation is amplified by PCR with the primer set UF and DR using pMKT-UPandDOWN as the template.

    1. PCR is carried out as described in Step A2 with the specific primer sets (Upstream Forward: UF and Downstream Reverse: DR) and pMKT-UPandDOWN as the template. 
    2. To digest the plasmid DNA, treat the amplified PCR mix with 10 units of DpnI at 37 °C for 1 h. 
    3. Purify the specific band using the Wizard SV Gel and PCR Clean-Up System after 1.2% agarose electrophoresis (applying a voltage of 100V for 30 min). Prepare 2-5 μg of DNA fragments for transformation in 40 μl of dH2O.

  5. Transformation of C. merolae cells
    Transform the C. merolae T1 cells following the polyethylene glycol-mediated protocol as described by Ohnuma et al. (2008) and Fujiwara and Ohnuma (2018).
    1. When OD750 reaches ~10, dilute the cell culture in 50 ml MA2 medium to yield an OD750 of 0.4 and cultivate in the glass cylinders (3 cm in diameter and 35 cm in length) with cotton plug bubbled with 2% CO2 in air under constant light (20 μmol photons m-2 s-1) at 40 °C for 20-24 h.
    2. Harvest the cells (OD750 < 0.7) by centrifugation (1,200 x g, for 5 min at 40 °C), and discard the supernatant. Resuspend the pellet with 200-300 μl of MA2 medium.
    3. Combine 2-5 μg of the DNA fragment in 40 μl of dH2O and 60 μl of DNA solution (see Recipes).
    4. Add 25 μl of the resuspended cells to the tube and mix gently.
    5. Add 125 μl of the PEG solution (see Recipes) immediately and mix the liquid by gently pipetting 4-5 times using the 200 μl pipette.
    6. Transfer the mixture to 30-40 ml of MA2 medium and incubate under the same condition as described in Step E1 for 20-24 h.
    7. Harvest the cultured cells by centrifugation (1,200 x g, for 5 min at 40 °C) and spread the cells onto the MA2 solid gellan gum plate without uracil and 5-FOA by the top starch method to increase the plating efficiency (Takemura et al., 2019). Incubate the plate at 40 °C supplemented with 5% CO2 in AnaeroPack with AnaeroPouch. 
    8. After 2-4 weeks, colonies will appear on the surface of the MA2 solid gellan gum plate.
    9. Pick up well-separated colonies (usually 20-24 candidates) using sterilized toothpicks, and inoculate 2 ml of MA2 liquid medium prepared in a 24-well tissue culture test plate.
    10. After incubation for 1-2 weeks in the same incubator without agitation, harvest the 20-24 candidate colonies and extract the DNA as described below (Kobayashi et al., 2010). To select the positive transformants, perform the PCR with the DNA using the ‘‘Confirmation primer sets’’ to anneal to the outside sequence of the homologous regions used to introduce the DNA fragment (Figure 3). PCR reactions are carried out using KOD-Plus-Neo DNA polymerase under the following reaction conditions: 94 °C for 2 min, followed by 30 cycles of 10 s at 98 °C, 30 s/1 kb at 68 °C, and a final extension of 2 min/1 kb at 68 °C.

  6. DNA extraction from C. merolae cells
    1. Harvest C. merolae cells by centrifugation at 3,000 x g for 5 min at 4 °C, and discard the medium.
    2. Resuspend the cell pellets with 400 μl of DNA extraction buffer [50 mM Tris-HCl (pH = 8.0), 5 mM EDTA, 0.5% Sodium dodecyl sulfate].
    3. Add 400 μl of neutralized phenol.
    4. Incubate at 65 °C for 10 min with shaking every 2 min by hand vigorously.
      Note: A vortex should not be used.
    5. Centrifuge at 12,000 x g for 5 min at room temperature. Transfer 375 μl of the above aqueous phase into a fresh tube, and add 375 μl of PCI (neutralized phenol:chloroform:isoamyl alcohol = 25:24:1). Invert the tube by hand vigorously.
    6. Centrifuge at 12,000 x g for 5 min at room temperature. Transfer 350 μl of the above aqueous phase into a fresh tube, and add 350 μl of PCI. Invert the tube by hand vigorously.
    7. Centrifuge at 12,000 x g for 5 min at room temperature. Transfer 300 μl of the above aqueous phase into a fresh tube, and add 30 μl of the 3 M sodium acetate and 750 μl of 100% ethanol. Invert the tube by hand vigorously.
    8. Centrifuge at 12,000 x g for 30 min at 4 °C. Discard the supernatant and add 200 μl of 75% ethanol.
    9. Centrifuge at 12,000 x g for 5 min at 4 °C. Discard the supernatant and let the liquid evaporate completely.
    10. Dissolve the precipitate of DNA in distilled water.
      Note: When using phenol-chloroform-isoamyl alcohol, personal protective equipment needs to be used according to local health and safety regulations. Always work under a fume hood.

  7. Elimination of URA5.3 gene marker from the genome
    1. The transformant cell using URA5.3T as a gene selectable marker is cultured in 50 ml of MA2 medium until the OD750 reaches 0.5-5.0.
    2. Centrifuge 50 ml of the culture at 1,200 x g for 5 min at 40 °C.
    3. Discard the supernatant, add 1.5 ml of MA2 medium and resuspend the precipitated cells.
    4. Add 1 ml of the top starch solution (Takemura et al., 2019) to 1 ml of the cell suspension, mix gently, and spread onto the MA2 solid gellan gum plate containing 0.5 mg/ml uracil and 0.8 mg/ml 5-FOA. 
    5. Add 1 ml of MA2 medium to the remaining cell suspension. Take 1 ml of the diluted cell suspension, and spread onto the MA2 solid gellan gum plate as in Step F4. 
    6. After 2-4 weeks, the colonies of the URA5.3 eliminated cells appear on the MA2 solid gellan gum plate.
    7. Pick up well-separated colonies (usually 20-24 candidates) using sterilized toothpicks, and inoculate each in 2 ml of MA2 liquid medium prepared in 24-well tissue culture test plate.
    8. After incubation for 1-2 weeks in the same incubator, extract DNA from each green colored inoculate as described in Step E10. To confirm URA5.3 elimination, perform the PCR with the DNA using the “Confirmation primer sets” to anneal to the outside sequence of the homologous regions used to introduce the DNA fragment as described in Step E10 (Figure 3).
    9. After confirmation of URA5.3 elimination by PCR, the strain is ready for the next transformation experiment using URA5.3T as a gene selectable marker.


      Figure 3. Schematic representation of confirmation of the transformation and URA5.3 elimination by PCR. A. Schematic representation of the modified gene loci of the host strain, 1st transformant and URA5.3 eliminated transformant. The arrows indicate the specific primer sets for confirmation of the transformation (named “Confirmation primer sets”). These primers should be designed to anneal to the outside sequence of the homologous regions. B. Example of gel electrophoresis image of PCR to confirm the expected recombination events using confirmation primer sets. 

Recipes

  1. DNA solution
    1. Mix 34 μl of dH2O and 6 μl of 10x MA2 solution I in a 1.5 ml microtube
    2. Add 20 μl of 10 mg/ml salmon sperm DNA to the above microtube
    3. Incubate the microtube at 40 °C until use
    Note: This solution should be prepared at time of use under a fume hood.
  2. PEG solution
    1. Take 0.6 g of polyethylene glycol 4000 in a 1.5 ml microtube
    2. Add 470 μl of 10x MA2 solution I to the microtube
    3. Incubate the microtube at 65 °C for 10 min shaking every 2 min by hand
    Note: This solution should be prepared at the time of use under a fume hood.
  3. MA2 medium
    1. Mix 100 ml of MA2 solution I, 10 ml of MA2 solution II, 1 ml of MA2 solution III and 885 ml of dH2O to a glass bottle and sterilize by autoclaving at 121 °C for 20 min
    2. Add 4 ml of MA2 solution IV to the mixture

      MA2 solution I


      A6 minor salts


      MA2 solution II


      MA2 solution III


      MA2 solution IV

      Note: This MA2 solution IV should be sterilized by filtration and stored at 4 °C in the dark.

  4. MA2 solid gellan gum plate (for making 9-10 plates)
    1. Mix 30 ml of MA2 solution I, 3 ml of MA2 solution II, 300 μl of MA2 solution III, and 70 ml of dH2O in a 500 ml flask. Mix 200 ml of dH2O and 1.5 g gellan gum in another 500 ml flask. Sterilize these flasks by autoclaving
    2. After autoclaving, mix each solution with 1.2 ml of MA2 solution IV in one flask. In case of 5-FOA selection, add directly 150 mg of uracil (final concentration: 0.5 mg/ml) and 240 mg of 5-FOA (final concentration: 0.8 mg/ml) in powder to the flask
    3. Pour the mixed solution onto the sterilized plastic plates
    4. Dry the plates at room temperature for 15 min
    5. If plates are to be stored, they should be kept in the dark at 4 °C
  5. DNA extraction buffer
    1. Dissolve 12.1 g of Trizma® base to 100 ml of dH2O in a glass bottle and adjust pH to 8.0 with HCl to make 1 M Tris-HCl (pH = 8.0) solution. Sterilize by autoclaving
    2. Dissolve 10.0 g of sodium dodecyl sulfate to 100 ml of dH2O in a glass bottle to make 10% SDS solution
    3. Dissolve 18.6 g of EDTA∙2Na to 100 ml of dH2O in a glass bottle and adjust pH to 8.0 with NaOH to make 0.5 M EDTA solution. Sterilize by autoclaving
    4. Mix 5 ml of 1 M Tris-HCl (pH = 8.0) solution, 5 ml of 10% SDS solution, 1 ml of 0.5 M EDTA solution and 89 ml of dH2O in a glass bottle
    5. Store at room temperature
  6. Neutralized phenol
    1. Dissolve 100 g of phenol in a 200 ml glass bottle by heating at 65 °C
    2. After dissolving phenol, add 0.1 g of 8-quinolinol to phenol
    3. Add the mixture of 50 ml of 1 M Tris-HCl (pH = 8.0) solution and 50 ml of dH2O to the phenol and mix well for 5 min at room temperature
    4. After mixing, incubate the phenol mixture for 10 min at room temperature
    5. Remove the 80 ml of the upper phase (liquid layer)
    6. Add the mixture of 50 ml of 1 M Tris-HCl (pH = 8.0) solution and 50 ml of dH2O to the phenol and mix well for 5 min at room temperature
    7. After mixing, incubate the phenol mixture for 10 min at room temperature
    8. Remove the 100 ml of the upper phase (liquid layer)
    9. Store at 4 °C in the dark
    Note: When using phenol-chloroform-isoamyl alcohol, personal protective equipment needs to be used according to local health and safety regulations. Always work under a fume hood.

Acknowledgments

This protocol was adapted from (Takemura et al., 2018 and 2019). The authors thank the Biomaterials Analysis Division, Tokyo Institute for Technology for DNA sequence analysis. This study was supported by MEXT/JSPS KAKENHI (Grant numbers: 15K14539 to K.T., 17K07438 to S.I., 17K07439 to Y.K.) and by Advanced Low Carbon Technology Research and Development Program (ALCA) of Japan Science and Technology Agency (JST) to K.T.

Competing interests

No competing interests declared.

References

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  6. Takemura, T., Imamura, S., Kobayashi, Y. and Tanaka, K. (2018). Construction of a selectable marker recycling system and the use in epitope tagging of multiple nuclear genes in the unicellular red alga Cyanidioschyzon merolae. Plant Cell Physiol 59(11): 2308-2316.
  7. Takemura, T., Imamura, S., Kobayashi, Y., and Tanaka, K. (2019). Top starch plating method for the efficient cultivation of unicellular red alga Cyanidioschyzon merolae. Bio-protocol 9(4): e3172.
  8. Taki, K., Sone, T., Kobayashi, Y., Watanabe, S., Imamura, S. and Tanaka, K. (2015). Construction of a URA5.3 deletion strain of the unicellular red alga Cyanidioschyzon merolae: A backgroundless host strain for transformation experiments. J Gen Appl Microbiol 61(5): 211-214.

简介

单细胞红藻 Cyanidioschyzon merolae 已被用作各种基础和应用研究的真核光合模型。 虽然 C的核基因组。 merolae 可以通过与外源导入的DNA的同源重组进行修饰,由于可用的阳性选择标记的数量有限,难以修饰同一菌株内的多个染色体位点。 最近,我们报道了一个修饰的 URA5.3 基因盒( URA5.3T ),可以使用pMKT质粒载体重复用于核基因组转化,用于表位标记(3x) FLAG-或3x Myc-)核编码蛋白质。 另外,这些质粒载体也可用于逐个敲除多个基因。 该报告描述了用于转化的DNA片段的构建和详细的转化程序。
【背景】 C中的标准转换方法。 merolae 使用尿嘧啶 - 营养缺陷型菌株M4(Minoda et al。,2004)或T1(Taki et al。,2015),它们带有移码或基因 URA5.3 ( CMK046C )中的完整缺失突变。基因 URA5.3 编码由乳清酸磷酸核糖基转移酶(OPRTase)结构域和乳清酸核苷-5'-磷酸脱羧酶(OMPdecase)结构域组成的蛋白质,该结构域从乳清酸合成尿苷-5'-一磷酸。通过将 URA5.3 作为选择标记引入尿嘧啶 - 营养缺陷型菌株中来选择转化体。最近的一项研究报道了氯霉素抗性 CAT 基因作为 C核转化实验的第二个选择标记基因。 merolae (Fujiwara et al。,2017)。然而,难以修饰超过三个基因组基因座。因此,我们使用改良的 URA5.3 基因开发了 URA5.3 选择标记基因回收系统,命名为 URA5.3T (Takemura 等人,2018)。 URA5.3T 包含 URA5.3 启动子 - 终止子盒5'端的 URA5.3 终止子序列(462 bp)进行同源重复,表位标签区编码三种不同蛋白酶(TEV,因子Xa和凝血酶)的识别序列,以及3x FLAG或3x Myc标签编码序列(图1)。通过位于 URA5.3T 两侧的同源重复序列经常发生的DNA切除,消除 URA5.3 启动子 - 终止子盒。在该方法中,使用5-氟乳清酸(5-FOA)选择 URA5.3 消除的转化体,其通过 URA5.3 活性转化为细胞毒性5-氟尿嘧啶。 。标记消除的转化体可以使用 URA5.3T 标记基因用于下一轮转化实验(图1)。在本报告中,我们提出了构建转化模板DNA的程序,其中包括回收标记 URA5.3T ,用于基因标记或基因敲除。


图1.使用 URA5.3T 从转化到 URA5.3 标记消除的概述。 DNA包括基因组特异性基因座的同源区域(灰色框)和 URA5.3T 。 URA5.3T 包括表位标签区,同源重复序列(红色阴影框)和 URA5.3 启动子 - 终止子盒。表位标签区含有三种不同蛋白酶(TEV,因子Xa和凝血酶)和3x FLAG或3x Myc标签编码序列的识别序列。

关键字:Cyanidioschyzon merolae, 表位标记, 基因敲除, 同源重组, 可筛选标记重复利用, URA5.3

材料和试剂

  1. 移液器吸头(NIPPON Genetics Co.,Ltd。,目录号:FG-102,FG-301,FG-401)
  2. 牙签
  3. Cuvettes(As One Corporation。,目录号:1-2855-02)
  4. 1.5毫升Microtube(BM Equipment Co.,Ltd。,目录号:NT-175)
  5. 50毫升离心管(Corning Incorporated,目录号:352196)
  6. 24孔组织培养试验板(BM Equipment Co.,Ltd。,目录号:92424)
  7. 灭菌塑料板(Φ90mm)(As One Corporation,目录号:GD90-15)
  8. AnaeroPouch(Mitsubishi Gas,目录号:2-3764-02)
  9. AnaeroPack(三菱燃气,目录号:2-3765-01)
  10. ℃。 merolae T1菌株(Taki et al。,2015)
  11. Escherichia coli DH5α(Takara Bio,目录号:9057)
  12. 野生型 C. merolae DNA
  13. 鲑鱼精子DNA(FUJIFILM Wako Pure Chemical Industries Corporation,目录号:047-17322)
  14. 质粒载体pMKT(Takemura et al。,2018;可根据要求提供给作者)
  15. KOD-Plus-Neo DNA聚合酶(Toyobo,目录号:KOD-401)
  16. GoTaq DNA聚合酶(Promega,目录号:M7123)
  17. Stu I(NEB,目录号:R0187S)
  18. Dpn I(Takara Bio,目录号:1235A)
  19. 特异性引物[用于扩增上游和下游片段的序列,用于 Stu I-消化的pMKT的同源序列(见图2A),在Integrated DNA Technologies(IDT)Inc。订购]
  20. 确认引物组(与同源区域的外部序列退火,参见图3,在Integrated DNA Technologies(IDT)Inc。订购)
  21. 用于菌落PCR的引物组(在Integrated DNA Technologies(IDT)Inc。订购):
    正向引物:5'-CGAGGTCGACGGTATCGATAAGC-3'
    反向引物:5'-GATCTAGTAACATAGATGACACCGC-3'
    正向引物下降:5'-GCTGCTAGGGATTGTGGCGCGA-3'
    向下反向引物:5'-CCGCTCTAGAACTAGTGGATCCC-3'
  22. In-fusion HD克隆试剂盒(Takara Bio,目录号:639648)
  23. 苯酚,晶体(FUJIFILM Wako Pure Chemical Corporation,目录号:169-22445)
  24. 氯仿(FUJIFILM Wako Pure Chemical Corporation,目录号:035-02616)
  25. 异戊醇(3-甲基-1-丁醇)(FUJIFILM Wako Pure Chemical Corporation,目录号:135-12015)
  26. Trizma ® base初级标准品和缓冲剂,结晶(Sigma-Aldrich,目录号:T1503-500G)
  27. HCl(FUJIFILM Wako Pure Chemical Corporation,目录号:081-03475)
  28. 十二烷基硫酸钠(SDS)(FUJIFILM Wako Pure Chemical Corporation,目录号:194-13985)
  29. NaOH(FUJIFILM Wako Pure Chemical Corporation,目录号:199-4895)
  30. 8-羟基喹啉(FUJIFILM Wako Pure Chemical Corporation,目录号:178-00752)
  31. 琼脂糖ME(岩井化学公司,目录号:50013R)
  32. Wizard SV凝胶和PCR清理系统(Promega,目录号:A9281)
  33. 聚乙二醇4000(FUJIFILM Wako Pure Chemical Corporation,目录号:5322-68-3)
  34. 玉米淀粉(Kawamitsu-Bussan,目录号:4901 486 02701 6)
  35. 顶级淀粉溶液(Takemura et al。,2019)
  36. 结冷胶(FUJIFILM Wako Pure Chemical Corporation,目录号:073-03071)
  37. Uracil(Sigma-Aldrich,目录号:U0750-5G)
  38. 5-氟乳清酸一水合物(5-FOA)(FUJIFILM Wako Pure Chemical Corporation,目录号:066-03663)
  39. 醋酸钠(FUJIFILM Wako Pure Chemical Corporation,目录号:190-01071)
  40. 乙醇(FUJIFILM Wako Pure Chemical Corporation,目录号:055-00457)
  41. (NH 4 ) 2 SO 4 (FUJIFILM Wako Pure Chemical Corporation,目录号:019-03435)
  42. MgSO 4 ∙7H 2 O(FUJIFILM Wako Pure Chemical Corporation,目录号:138-00415)
  43. H 2 SO 4 (FUJIFILM Wako Pure Chemical Corporation,目录号:192-04696)
  44. H 3 BO 3 (FUJIFILM Wako Pure Chemical Corporation,目录号:021-02195)
  45. MnCl 2 ∙4H 2 O(FUJIFILM Wako Pure Chemical Corporation,目录号:133-00725)
  46. ZnCl 2 (FUJIFILM Wako Pure Chemical Corporation,目录号:268-01022)
  47. Na 2 MoO 4 ∙2H 2 O(FUJIFILM Wako Pure Chemical Corporation,目录号:198-02471)
  48. CoCl 2 ∙6H 2 O(FUJIFILM Wako Pure Chemical Corporation,目录号:038-03681)
  49. CuCl 2 ∙2H 2 O(FUJIFILM Wako Pure Chemical Corporation,目录号:039-04135)
  50. KH 2 PO 4 (FUJIFILM Wako Pure Chemical Corporation,目录号:169-04245)
  51. CaCl 2 ∙2H 2 O(FUJIFILM Wako Pure Chemical Corporation,目录号:038-19735)
  52. FeCl 3 ∙6H 2 O(FUJIFILM Wako Pure Chemical Corporation,目录号:090-02802)
  53. Na 2 EDTA(FUJIFILM Wako Pure Chemical Corporation,目录号:345-01865)
  54. DNA解决方案(见食谱)
  55. PEG解决方案(见食谱)
  56. MA2培养基(Kobayashi et al。,2010,见食谱)
    1. MA2解决方案I
    2. A6次盐
    3. MA2解决方案II
    4. MA2解决方案III
    5. MA2解决方案IV
  57. MA2固体结冷胶板(见食谱)
  58. DNA提取缓冲液(见食谱)
  59. 中和酚(见食谱)

设备

  1. 移液器(Gilson,Inc.,目录号:F123600,F123601,F123602)
  2. 冷冻离心机(Koki Holdings Co.,Ltd.,型号:CF16RXII)
  3. 微量离心机(TOMY Seiko Co.,Ltd。,型号:MX150)
  4. 500毫升烧瓶(IWAI,目录号:4442FK500)
  5. 热块(千代田科技有限公司,型号:MiniT-100)
  6. 生物培养箱(TOMY Seiko Co.,Ltd.,型号:CLE-303)
  7. NanoDrop(Thermo Fisher Scientific Inc.,型号:ND-LITE)
  8. 热循环仪(Bio-Rad Laboratories,Inc。,型号:T100热循环仪)
  9. 电泳仪(Atto Corporation,型号:WSE-1710)
  10. FAS凝胶成像仪(Nippon Genetics Co.,Ltd。,型号:FAS-IV)
  11. 分光光度计(Beckman coulter,Inc。,型号:DU730,A23616)

程序

  1. 构建引入的DNA片段
    构建概述:PCR扩增的DNA片段(基因组特异性基因座的同源区域的上游和下游)和 Stu I消化的pMKT(包括两个片段消化的载体L和S)使用In-fusion系统生成pMKT-UPandDOWN。所得质粒用作模板DNA,用于通过PCR扩增引入的DNA片段(图2)。
    DNA片段的PCR扩增(图2A):
    1. 如下给出设计特异性引物组以扩增DNA片段(每个500-1,500bp)用于同源重组至基因组特异性基因座。上游和下游DNA片段不需要共享相同的长度。对于标记,上游DNA片段应包括缺乏终止密码子的开放阅读框,其与框架标签框内融合。每个有色区域(20bp)是相关末端的同源序列 Stu I-消化的pMKT(颜色对应于图2A)和每个重复的'Ns'(20-25bp)表示退火序列以扩增上游和下游DNA片段。每个引物的退火序列('Ns')的T m 应设计为高于63°C。
      上游前进(UF):
      5' - GACACGTGAATTTAAATAGG NNNNNNNNNNNNNNNNNNNN-3'
      上游反向(UR):
      5' - TGAAAATAAAGATTTTCAGG NNNNNNNNNNNNNNNNNNNN-3'
      下游前进(DF):
      5' - AGGGATTGTGGCGCGACAGG NNNNNNNNNNNNNNNNNNNN-3'
      下游反向(DR):
      5' - GACACGTGAATTTAAATAGG NNNNNNNNNNNNNNNNNNNN-3'
    2. 使用KOD-Plus-Neo DNA聚合酶在以下反应条件下进行PCR反应:94℃持续2分钟,然后进行30个循环:在98℃下10秒,在68℃下30秒/ 1kb,和使用特异性引物组(上游的UF / UR和下游的DF / DR)和50ng野生型 C在68℃下最终延伸2分钟/ 1kb。 merolae DNA作为模板。使用Wizard SV Gel和PCR Clean-Up System按照制造商的方案纯化PCR扩增的DNA片段,并测量DNA的浓度。
      注意:我们通常通过上述条件获得可重复的扩增结果。应通过凝胶电泳检查PCR结果以确认扩增。

  2. 通过 Stu I进行pMKT载体消化以获得两个DNA片段,这里称为消化载体L(长,约3.6kbp)和S(短,约3.0kbp,图2B)
    1. 将1μgpMKTDNA在含有10单位 Stu I的50μl1xCutSmart缓冲液(即,提供 Stu I缓冲液)中孵育37 °C 1小时。
    2. 加入100μldH 2 O和150μl苯酚:氯仿:异戊醇= 25:24:1溶液并剧烈混合。在室温下以12,000 x g 离心10分钟。取150μl上清液,加入15μl3M乙酸钠和375μl100%EtOH。剧烈混合并在4,000℃下以12,000 x g 离心30分钟。弃去上清液,向沉淀中加入75%EtOH,并在4℃下以12,000 x g 离心5分钟。弃去上清液,在冲洗离心后用移液管完全除去剩余的液滴,并在室温下在工作台上短暂风干颗粒。然后加入20μldH 2 O并溶解沉淀的DNA。测量DNA浓度。
      注意:使用苯酚 - 氯仿 - 异戊醇时,需要根据当地的健康和安全法规使用个人防护设备。总是在通风橱下工作。

  3. 融合反应产生模板质粒(图2C)
    1. 轻轻混合5μlDNA混合物[ Stu I消化的pMKT(消化的载体L和S):上游:下游≈100fmol:50 fmol:50 fmol]和5μl2xIn-fusion反应混合。将反应混合物在50℃孵育15分钟,然后在冰上冷却。
    2. 用常规使用的化学转化方法用反应混合物转化大肠杆菌DH5α。通过菌落PCR找到预期的质粒构建体。使用GoTaq DNA聚合酶在以下反应条件下进行PCR反应:95℃持续3分钟,然后进行30个循环:在95℃下30秒,在55℃下30秒,在72℃下1分钟/ 1kb。用引物组在72℃最终延伸2分钟(向前引物:5'-CGAGGTCGACGGTATCGATAAGC-3'/反向引物用于上游:5'-GATCTAGTAACATAGATGACACCGC-3'用于上游序列,和正向引物用于向下:5'-GCTGCTAGGGATTGTGGCGCGA-3'/用于向下的反向引物:用于下游序列的5'-CCGCTCTAGAACTAGTGGATCCC-3'。随后,通过用于菌落PCR的引物组进行DNA序列分析来检查阳性候选物。&nbsp;
    3. 提取并纯化质粒DNA(pMKT-UPandDOWN)。

  4. 通过PCR扩增DNA进行转化以制备线性化DNA片段并完全去除模板环状质粒DNA(图2D)
    注意:经常包含模板质粒DNA可能导致质粒随机整合到基因组中。因此,应小心移除模板质粒DNA。


    图2.模板载体(pMKT-UPandDOWN)和用于转化的DNA构建体的概述。 A.对应于靶基因的DNA区域(上游和下游)通过PCR扩增,具有特定的引物组pMKT的20bp同源序列(显示为红色,蓝色,绿色和黄色)。 B.用 Stu I酶消化pMKT载体,在其长度(L-long; S-short)后产生2个名为消化的载体L和S的DNA片段。红色阴影区域表示同源重复序列(,即URA5.3 终止子区域)。 C.使用In-融合系统将四个DNA片段(消化的载体L,消化的载体S,上游和下游)连接在一个反应管中,以制备质粒pMKT-UP和DOWN)。 D.使用pMKT-UPandDOWN作为模板,用引物组UF和DR通过PCR扩增用于转化的DNA片段。

    1. 如步骤A2中所述进行PCR,其中特异性引物组(上游前向:UF和下游反向:DR)和pMKT-UPandDOWN作为模板。&nbsp;
    2. 为了消化质粒DNA,将扩增的PCR混合物用10单位的 Dpn I在37°C处理1小时。&nbsp;
    3. 在1.2%琼脂糖电泳(施加100V电压30分钟)后,使用Wizard SV Gel和PCR Clean-Up System纯化特异性条带。制备2-5μgDNA片段,用于在40μldH 2 O中转化。

  5. C的转换。 merolae 细胞
    转换 C。按照Ohnuma 等人(2008)和Fujiwara和Ohnuma(2018)所述的聚乙二醇介导的方案遵循merolae T1细胞。
    1. 当OD 750 达到~10时,将细胞培养物稀释在50ml MA2培养基中,得到OD 750 0.4,并在玻璃圆筒中培养(直径3cm,35在恒定光照下,在空气中用2%CO 2 鼓泡的棉塞(20μmol光子m -2 s -1 )在40°C下保持20-24小时。
    2. 通过离心(1,200 x g ,在40℃下5分钟)收获细胞(OD 750 <0.7),并弃去上清液。用200-300μlMA2培养基重悬沉淀。
    3. 将2-5μgDNA片段在40μldH 2 O和60μlDNA溶液中混合(参见配方)。
    4. 将25μl重悬浮的细胞加入管中并轻轻混合。
    5. 立即加入125μlPEG溶液(参见食谱),用200μl移液管轻轻移液4-5次,混合液体。
    6. 将混合物转移至30-40ml MA2培养基中,并在与步骤E1中所述相同的条件下孵育20-24小时。
    7. 通过离心(1,200 xg ,在40℃下5分钟)收获培养的细胞,并通过顶部淀粉方法将细胞铺展到不含尿嘧啶和5-FOA的MA2固体结冷胶板上以增加电镀效率(Takemura et al。,2019)。在AnaeroPack中使用AnaeroPouch在40°C下补充5%CO 2 孵育平板。&nbsp;
    8. 2-4周后,菌落将出现在MA2固体结冷胶板的表面上。
    9. 使用灭菌牙签挑取分离良好的菌落(通常20-24个候选菌株),并接种在24孔组织培养试验板中制备的2ml MA2液体培养基。
    10. 在不搅拌的情况下在相同的培养箱中孵育1-2周后,收获20-24个候选菌落并如下所述提取DNA(Kobayashi 等人,,2010)。为了选择阳性转化体,使用''确认引物组'用DNA进行PCR以退火到用于引入DNA片段的同源区域的外部序列(图3)。使用KOD-Plus-Neo DNA聚合酶在以下反应条件下进行PCR反应:94℃持续2分钟,然后进行30个循环:在98℃下10秒,在68℃下30秒/ 1kb,和在68°C下最终延伸2分钟/ 1 kb。

  6. 从 C中提取DNA。 merolae 细胞
    1. 收获 C.通过在4℃下以3,000μL离心5分钟离心5分钟的merolae 细胞,并丢弃培养基。
    2. 用400μlDNA提取缓冲液[50mM Tris-HCl(pH = 8.0),5mM EDTA,0.5%十二烷基硫酸钠]重悬细胞沉淀。
    3. 加入400μl中和的苯酚。
    4. 在65°C孵育10分钟,每2分钟用手剧烈摇动。
      注意:不应使用漩涡。
    5. 在室温下以12,000 x g 离心5分钟。将375μl上述水相转移到新管中,并加入375μlPCI(中和的苯酚:氯仿:异戊醇= 25:24:1)。用力手动翻转管子。
    6. 在室温下以12,000 x g 离心5分钟。将350μl上述水相转移到新管中,并加入350μlPCI。用力手动翻转管子。
    7. 在室温下以12,000 x g 离心5分钟。将300μl上述水相转移到新管中,并加入30μl3M乙酸钠和750μl100%乙醇。用力手动翻转管子。
    8. 在4℃下以12,000 x g 离心30分钟。弃上清,加入200μl75%乙醇。
    9. 在12,000 x g 下在4℃下离心5分钟。弃去上清液,让液体完全蒸发。
    10. 将DNA沉淀溶解在蒸馏水中。
      注意:使用苯酚 - 氯仿 - 异戊醇时,需要根据当地的健康和安全法规使用个人防护设备。总是在通风橱下工作。

  7. 从基因组中消除 URA5.3 基因标记
    1. 使用 URA5.3T 作为基因选择标记的转化体细胞在50ml MA2培养基中培养直至OD 750 达到0.5-5.0。
    2. 将离心的50ml培养物在1200℃下在40℃下离心5分钟。
    3. 弃去上清液,加入1.5ml MA2培养基并重悬沉淀的细胞。
    4. 将1ml顶部淀粉溶液(Takemura et al。,2019)加入1ml细胞悬浮液中,轻轻混合,涂在含有0.5mg / ml尿嘧啶的MA2固体结冷胶板上。 0.8 mg / ml 5-FOA。&nbsp;
    5. 向剩余的细胞悬浮液中加入1ml MA2培养基。取1 ml稀释的细胞悬浮液,按步骤F4涂抹在MA2固体结冷胶板上。&nbsp;
    6. 2-4周后, URA5.3 的菌落消除细胞出现在MA2固体结冷胶板上。
    7. 使用灭菌牙签挑取分离良好的菌落(通常为20-24个候选菌),并将其各自接种在24孔组织培养试验板中制备的2ml MA2液体培养基中。
    8. 在相同的培养箱中孵育1-2周后,如步骤E10所述从每个绿色接种物中提取DNA。以确认 URA5.3 消除,如步骤E10所述的使用所述“确认引物组”退火至用于引入DNA片段的同源区的外侧序列的DNA进行PCR(图3)。
    9. 确认后的 URA5.3 消除通过PCR,使用菌株是准备进行下一次转化实验 URA5.3T 作为基因选择标记。


      的图3中的变换的确认的示意图和 的 URA5.3 的消除由PCR A.示意图对于宿主菌株的修饰基因座,第一转化体和 URA5.3 消除了转化体。箭头表示用于确认转化的特异性引物组(命名为“确认引物组”)。应设计这些引物以与同源区域的外部序列退火。 B.使用确认引物组确认预期重组事件的PCR凝胶电泳图像的实例。&nbsp;

食谱

  1. DNA解决方案
    1. 在1.5 ml微管中混合34μldH 2 O和6μl10xMA2溶液I
    2. 向上述微管中加入20μl10mg/ ml鲑鱼精子DNA
    3. 将微管在40°C孵育直至使用
    注意:此溶液应在通风橱下使用时准备好。
  2. PEG解决方案
    1. 在1.5ml微管中取0.6g聚乙二醇4000
    2. 向微管中加入470μl10xMA2溶液I.
    3. 将微管在65℃下孵育10分钟,每2分钟用手摇动一次
    注意:此溶液应在通风橱下使用时准备好。
  3. MA2培养基
    1. 将100ml MA2溶液I,10ml MA2溶液II,1ml MA2溶液III和885ml dH 2 O混合到玻璃瓶中,并通过在121℃高压灭菌20分钟进行灭菌。
    2. 向混合物中加入4ml MA2溶液IV
      MA2解决方案我


      A6次盐


      MA2解决方案II


      MA2解决方案III


      MA2解决方案IV

      注意:此MA2溶液IV应通过过滤灭菌,并在4°C黑暗中储存。

  4. MA2固体结冷胶板(用于制作9-10个板)
    1. 在500ml烧瓶中混合30ml MA2溶液I,3ml MA2溶液II,300μlMA2溶液III和70ml dH2S O.在另一个500ml烧瓶中混合200ml dH 2 O和1.5g结冷胶。通过高压灭菌消毒这些烧瓶
    2. 高压灭菌后,将每种溶液与1.2ml MA2溶液IV在一个烧瓶中混合。在5-FOA选择的情况下,直接向烧瓶中加入150毫克尿嘧啶(终浓度:0.5毫克/毫升)和240毫克5-FOA(终浓度:0.8毫克/毫升)粉末
    3. 将混合溶液倒入灭菌的塑料板上
    4. 在室温下干燥板15分钟
    5. 如果要储存板,则应将它们保存在4°C的黑暗中
  5. DNA提取缓冲液
    1. 将12.1克Trizma ®碱溶于玻璃瓶中的100毫升dH 2 O中,用HCl调节pH至8.0,制成1M Tris-HCl(pH = 8.0) )解决方案。通过高压灭菌消毒
    2. 将10.0g十二烷基硫酸钠溶于玻璃瓶中的100ml dH 2 O中,制成10%SDS溶液
    3. 将18.6g EDTA∙2Na溶解在玻璃瓶中的100ml dH 2 O中并用NaOH调节pH至8.0以制备0.5M EDTA溶液。通过高压灭菌消毒
    4. 在玻璃瓶中混合5毫升1M Tris-HCl(pH = 8.0)溶液,5毫升10%SDS溶液,1毫升0.5M EDTA溶液和89毫升dH 2 O
    5. 在室温下储存
  6. 中和的苯酚
    1. 通过在65℃加热将100g苯酚溶解在200ml玻璃瓶中
    2. 溶解苯酚后,向苯酚中加入0.1g 8-羟基喹啉
    3. 将50ml 1M Tris-HCl(pH = 8.0)溶液和50ml dH 2 O的混合物加入到苯酚中并在室温下充分混合5分钟
    4. 混合后,在室温下孵育酚混合物10分钟
    5. 去除80毫升上层相(液体层)
    6. 将50ml 1M Tris-HCl(pH = 8.0)溶液和50ml dH 2 O的混合物加入到苯酚中并在室温下充分混合5分钟
    7. 混合后,在室温下孵育酚混合物10分钟
    8. 去除100毫升上层相(液体层)
    9. 在黑暗中于4°C储存
    注意:使用苯酚 - 氯仿 - 异戊醇时,需要根据当地的健康和安全法规使用个人防护设备。总是在通风橱下工作。

致谢

该方案改编自(Takemura et al。,2018和2019)。作者感谢东京工业大学DNA序列分析生物材料分析部门。该研究得到MEXT / JSPS KAKENHI(授权号:15K14539至K.T.,17K07438至S.I.,17K07439至Y.K.)和日本科学技术厅(JST)的先进低碳技术研究与开发计划(ALCA)至K.T.的支持。

利益争夺

没有宣布竞争利益。

参考

  1. Fujiwara,T.,Ohnuma,M.,Kuroiwa,T.,Ohbayashi,R.,Hirooka,S。和Miyagishima,S。Y.(2017)。 基于新建立的氯霉素,通过两步转化开发双核基因靶向方法 - 红藻中的选择系统 Cyanidioschyzon merolae 。 Front Plant Sci 8:343。
  2. Kobayashi,Y.,Ohnuma,M.,Kuroiwa,T.,Tanaka,K。和Hanaoka,M。(2010)单细胞红藻 Cyanidioschyzon merolae 的培养和分子遗传分析的基础。 Endocytobiosis Cell Res 20:53-61。
  3. Minoda,A.,Sakagami,R.,Yagisawa,F.,Kuroiwa,T。和Tanaka,K。(2004)。 通过红藻中的同源重组改善培养条件和核转化证据, Cyanidioschyzon merolae 10D。 植物细胞生理学 45(6):667-671。
  4. Fujiwara,T。和Ohnuma,M。(2018)。 转换程序及其在 Cyanidioschyzon merolae中的应用
  5. Ohnuma,M.,Yokoyama,T.,Inouye,T.,Sekine,Y。和Tanaka,K。(2008)。 聚乙二醇(PEG)介导的红藻中的瞬时基因表达, Cyanidioschyzon merolae 10D。 植物细胞生理学 49(1):117-120。
  6. Takemura,T.,Imamura,S.,Kobayashi,Y。和Tanaka,K。(2018)。 选择性标记物回收系统的构建以及单细胞红中多个核基因的表位标记的用途alga Cyanidioschyzon merolae 。 植物细胞生理学 59(11):2308-2316。
  7. Takemura,T.,Imamura,S.,Kobayashi,Y。和Tanaka,K。(2019)。 用于有效培养单细胞红藻 Cyanidioschyzon merolae 的顶级淀粉接种方法。 生物协议 9(4):e3172。
  8. Taki,K.,Sone,T.,Kobayashi,Y.,Watanabe,S.,Imamura,S。和Tanaka,K。(2015)。 构建单细胞红藻的 URA5.3 缺失株 Cyanidioschyzon merolae :用于转化实验的无背景宿主菌株。 J Gen Appl Microbiol 61(5):211-214。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Takemura, T., Imamura, S., Kobayashi, Y. and Tanaka, K. (2019). Multiple Modification of Chromosomal Loci Using URA5.3 Selection Marker in the Unicellular Red Alga Cyanidioschyzon merolae. Bio-protocol 9(7): e3204. DOI: 10.21769/BioProtoc.3204.
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