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Transformation of the Cyanobacterium Leptolyngbya boryana by Electroporation
电击法转化蓝藻鞘丝藻   

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Abstract

Leptolyngbya boryana (L. boryana) (formerly Plectonema boryanum) is a versatile, filamentous cyanobacterium that has the ability to fix nitrogen under microoxic conditions and to grow heterotrophically with glucose in the dark, providing an excellent system to investigate photosynthesis, nitrogen fixation, and their regulatory mechanisms. While L. boryana is not naturally transformable different from the unicellular cyanobacterium Synechocystis sp. PCC 6803, it can be transformed by electroporation. Here we describe the transformation of L. boryana by electroporation to isolate mutants in which a targeted gene is disrupted.

Keywords: Cyanobacteria(蓝藻), Transformation(转型), Electroporation(电穿孔), Targeted gene disruption(靶向基因中断), Shuttle vector(穿梭载体)

Materials and Reagents

  1. Bottle top filter system (0.22 µm) (Corning, catalog number: 430624 )
  2. Microcentrifuge tubes (1.5 ml) (Ina-optika corporation, BIO-BIK, catalog number: ST-0150F ) or its equivalent, sterilized by autoclave (121 ºC, 20 min)
  3. Micropipettes tips (121 ºC, 20 min)
  4. Sterile syringe filter (Millex-GV Syringe Filter Unit, 0.22 µm), used for filter sterilization of solutions of antibiotics and glucose (Thermo Fisher Scientific, Millipore, catalog number: SLGV033RV ) or its equivalent
  5. Petri dish [sterile Petri dishes (90 mm x 15 mm)] (ASONE Corporation, catalog number: 1-7484-01-30 ) or its equivalent
  6. Pulse cuvettes (Gene Pulser cuvette, 0.1 cm) (Bio-Rad Laboratories, AbD Serotec®, catalog number: 1652089 )
  7. Hybond N+ filter (disc 82 mm diameter) (GE Healthcare, Amersham, catalog number: RPN82B ), sterilized by autoclave (121 ºC, 20 min).
  8. Leptolyngbya boryana (wild type or dg5) (grown on a BG-11 agar plate supplemented with 20 mM HEPES-KOH, pH 7.5 and 30 mM glucose)
    Note: The dg5 strain was isolated from the wild type as a natural mutant that grows much faster heterotrophically in the dark than wild type (Fujita et al., 1996). Recently we identified that the mutation responsible for the dg5 phenotype is one adenine insertion causing a frameshift in the cytM gene encoding cytochrome cM (Hiraide et al., 2015).
  9. Sterilized water (150 ml)
    Note: Water is purified by RiOs Essential Water Purification System (Merck Millipore Corporation) or WEX system (Yamato). Distilled water can be also used. Autoclave (121 ºC, 20 min) and chill on ice before use.
  10. NaNO3 (Nacalai tesque, catalog number: 31617-35 )
  11. 1 M K2HPO4 solution (Wako Pure Chemical Industries, Siyaku, catalog number: 164-04295 )
  12. 7.5% (w/v) MgSO4.7H2O (Nacalai tesque, catalog number: 21003-75 )
  13. 3.6% (w/v) CaCl2.2H2O (Nacalai tesque, catalog number: 06731-05 )
  14. 2.0% (w/v) Na2CO3 (Nacalai tesque, catalog number: 31311-25 )
  15. Citric acid (Wako Pure Chemical Industries, Siyaku, catalog number: 038-05521 )
  16. Ammonium iron (III) citrate, brown (Wako Pure Chemical Industries, Siyaku, catalog number: 092-00802 )
  17. Ethylenediamine-N, N, N', N'-tetraacetic acid, disodium salt, dihydrate (EDTA-Na2) (Dojindo, catalog number: 345-01865 )
  18. H3BO4 (Wako Pure Chemical Industries, Siyaku, catalog number: 021-02195 )
  19. MnCl2.4H2O (Nacalai tesque, catalog number: 21211-45 )
  20. ZnSO4.7H2O (Wako Pure Chemical Industries, Siyaku, catalog number: 268-00405 )
  21. Na2MoO4.2H2O (Wako Pure Chemical Industries, Siyaku, catalog number: 197-02485 )
  22. CuSO4.5H2O (Wako Pure Chemical Industries, Siyaku, catalog number: 033-04415 )
  23. Co(NO3)2.6H2O (Wako Pure Chemical Industries, Siyaku, catalog number: 031-03752 )
  24. 1 M Glucose (filter sterilized) (Wako Pure Chemical Industries, Siyaku, catalog number: 049-31165 )
  25. 50 mg/ml kanamycin sulfate (filter sterilized) (Wako Pure Chemical Industries, Siyaku, catalog number: 111-00344 )
  26. 50 mg/ml chloramphenicol (dissolve in ethanol) (Nacalai tesque, catalog number: 08027-72 )
  27. 10 mg/ml streptomycin sulfate (filter sterilized) (Meiji Seika Pharma, catalog number: 4987222665643 )
  28. 50 mg/ml erythromycin (filter sterilized) (Wako Pure Chemical Industries, Siyaku, catalog number: 054-05101 )
  29. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Nacalai tesque, catalog number: 17514-15 )
  30. Plasmid preparation kit (GenElute HP Plasmid Midiprep Kit) (Sigma-Aldrich, catalog number: NA0200 )
  31. Linearized plasmid solution (> 2 µg µl–1)
    Note: The concentration of plasmid is determined by a spectrophotometer before the enzyme digestion.
  32. Bacto Agar [1.5% (w/v)] (BD, catalog number: 214010 )
  33. Ferric citrate solution (see Recipes)
  34. Trace metal A5+Co solution (1 L) (see Recipes)
  35. 2x BG-11 solution (1 L) (see Recipes)
  36. BG-11 agar plates (supplemented with 20 mM HEPES-KOH, pH 7.5, with or without appropriate antibiotics, Table 1) solidified with Bacto Agar [1.5% (w/v)] (BD, catalog number: 214010) (see Recipes)

Equipment

  1. Forceps
  2. Micropipettes ( P-1000 , P-200 and P-20 , or their equivalents) (Gilson)
  3. Clean bench
  4. Autoclave (TOMY DIGITAL BIOLOGY CO., model: SX-500 or its equivalent)
  5. Gene pulser Xcell (Bio-Rad Laboratories, AbD Serotec®, catalog numbers: 1652666 , 1652668 and 1652669 )
  6. Spectrophotometer (Shimadzu, model: UV-1700 or GE Healthcare, model: NanoVue Plus or its equivalent)
  7. Aspirator (with a vacuum pump)
  8. Growth cabinet (with appropriate fluorescence light bulbs)

Procedure

  1. Plasmid for targeted mutagenesis is constructed (Figure 1). Plasmids carry a chimeric DNA fragment consisting of the upstream sequence of the target gene, drug resistance cartridge (Table 1), and downstream sequence of the target gene (Figure 1B). The upstream and downstream sequences should be more than 1 kb each, for stable homologous recombination. If the upstream and downstream fragments are obtained by PCR, their nucleotide sequences in the plasmid should be confirmed by Sanger sequencing to avoid introduction of unintended mutation(s) outside of the target gene via homologous recombination. The vectors are normally pUC18/19 derivatives.


    Figure 1. An example of plasmid construction, homologous recombination and PCR analysis of mutants in L. boryana. A. Gene arrangement of the target gene (the orf84 gene, LBWT_23230, shown in red) in L. boryana (Tsujimoto et al., 2014). The 1.7-kb upstream and 1.5-kb downstream fragments (thick horizontal bars) of the orf84 gene are amplified by PCR to construct plasmid. B. Construction of the plasmid pNK72 to isolate an orf84-disrupted mutant (∆orf84). The two amplified fragments were cloned into a vector. In this case, pUC192 was used as the vector, which was constructed by the insertion of the neo gene (kanamycin resistance gene from pMC19, Fujita et al., 1992) into the BamHI site of pUC19 (dashed line with its multi-cloning sites shown by yellow boxes). The 1.5-kb downstream fragment (digested with SalI-XhoI) was cloned into the SalI sites of pUC192, and subsequently the 1.7-kb upstream fragment (digested with BamHI) was cloned into the BamHI site, yielding the plasmid pNK72. C. Gene arrangement of a single recombinant, in which the plasmid pNK72 is incorporated into the chromosome by a single homologous recombination event. Both wild-type and knock-out copies are present in the chromosome. In this case the single recombination event occurs between the 1.5-kb downstream fragment of the plasmid and the corresponding chromosomal part. Small horizontal arrows indicate primers for PCR shown in panel E. D. Gene arrangement of the ∆orf84 mutant, in which the orf84 gene was replaced with the neo gene by a double recombination event. The plasmid was previously digested with KpnI and SalI for electroporation to avoid to generate single recombinants. Small horizontal arrows indicate primers for PCR shown in panel F. E. PCR analysis of the single recombinants of orf84. Two DNA fragments were detected. The longer (a filled triangle, 1,799 bp) and shorter (an open triangle, 437 bp) fragments are corresponding to those of knock-out and wild-type copies, respectively, in the three transformants (lanes 3-5). The longer fragment was detected in PCR with the plasmid pNK72 as the template (lane 2). The shorter fragment (the wild-type copy) was detected in the wild-type cells (lane 1). F. PCR analysis of ∆orf84. Only single DNA fragment corresponding to the knock-out copy of orf84 (a filled triangle, 1,799 bp) was detected in the two transformants (lanes 3 and 4). The shorter and longer fragments (lanes 1 and 2) were the same as E. (unpublished results, Kotani and Fujita).

  2. The plasmid prepared by a plasmid preparation kit was linearized by restriction enzyme(s) to minimize the probability that the plasmid would integrate into the genome via a single recombination event resulting in a merodiploid that harbors both knock-out and wild-type copies in the genome (Figure 1C). The digestion site(s) can be selected from anywhere in the vector part. It should be careful not to digest the parts of homologous recombination and the drug resistance marker. The digested plasmid was precipitated by ethanol and dissolved in sterilized water to be >2 µg µl–1.
  3. L. boryana is cultivated on an agar plate of BG-11 containing 30 mM glucose under low light conditions (<10 µmol m-2 s-1) at 30°C for 2-7 days (Note 1).
  4. All procedures below should be performed under aseptic conditions. Cells on the agar plate are suspended in sterilized water (ca. 2 ml per one agar plate) and the suspension is transferred into a bottle top filter (Figure 2A-C. Notes 2 and 3). In this stage, Hybond N+ filters should be placed onto new BG-11 agar plates for cell recovery after electroporation (step 11).
  5. The cells are collected on the surface of the bottle top filter by aspiration (Figure 2D).


    Figure 2. Preparation of L. boryana cells for pulse application

  6. Approximately 50 ml of sterilized water, chilled on ice, is added to the filter to suspend the cells. The cells are again collected on the filter by aspiration. This washing procedure is repeated three times (Figure 3C. Notes 4-5). Centrifugation (1,200 x g, 10 min) is another option for washing cells with water. In this case, note that L. boryana cells form very loose pellets even by high-speed centrifugation (for example, 44,000 x g, 30 min) due to long trichomes.
  7. The cells on the filter are suspended in a small aliquot (500 µl) of sterilized water (Figure 2D) and the suspension is transferred into a microcentrifuge tube (1.5 ml) to store on ice until pulse application (Note 3).
  8. A small aliquot (50 µl) of the suspension used for pulse application is dispensed into another microcentrifuge tube (Note 3) and the concentrated plasmid solution (typically 10 µl of 2 µg µl-1) is added to the tube and mixed well.
  9. The mixture is transferred into a cuvette with a gap width of 0.1 cm and chilled on ice (Figure 2E-F, Note 3).
  10. A single exponential decay pulse (setting: voltage, 1.41 kV; capacitance, 50 µF; and resistance, 250 ohms) is applied. The time constant will be approximately 10 ms. The time constant and the actual voltage should be recorded on your note (Notes 5-6).
  11. An aliquot (350 µl) of BG-11 liquid medium is added to the cuvette and the cell suspension is recovered with the blue tip of a P-1000 type micropipette dispenser (Figure 3A) to spread onto a Hybond N+ filter overlaid on a BG-11 agar plate without antibiotics (Figure 3B-C).


    Figure 3. Recovery of pulse-applied cells from a cuvette and inoculation on a non-selective plate

  12. The agar plate is incubated at 30 °C under low light conditions (<10 µmol m-2 s-1) for 2 days. During this period the antibiotic resistance gene is expressed to confer the cell’s resistance to the specific antibiotic.
  13. The Hybond N+ filer is transferred onto a new BG-11 agar plate containing appropriate antibiotics (final concentrations are shown in Table 1). The agar plate is incubated at 30 °C under high light conditions (ca. 100 µmol m-2 s-1).
  14. Colonies showing resistance against the antibiotics appear after approximately 10 to 14 days (Figure 4). You can pick them up with toothpicks or tips (for a P-200-type micropipette) that should be previously autoclaved (121 °C, 20 min).
  15. Some colonies are single recombinants and some are double recombinants. PCR analysis should be carried out to identify double recombinants (Figure 1E-F; Notes 7 -8).

    Table 1. List of antibiotics that can be used for transformation of L. boryana

    1This antibiotic has not yet been used for targeted mutagenesis. However, we confirmed that transformants harboring pJRD215 (with the streptomycin resistance gene) showed resistance to streptomycin (unpublished results).

Representative data

Representative results in targeted gene disruption are shown in Table 2. In most cases almost all transformants appeared on selective plates were double recombinants. A procedure of repeated inoculations of transformants to segregate cells in which all wild-type copies are completely replaced with the mutant copies is required in Synechocystis sp. PCC 6803, but this process is not needed in L. boryana (Note 8).

Table 2. Examples of electroporation of L. boryana

1
WT, wild type
2
Total DNA amount in the cell suspension for pulse application
3
Number of colonies. Single or double recombinant was confirmed by Southern blot analysis or PCR

Figure 4. Typical appearance of transformants on a selective plate. Two tiny green colonies (red arrows) appeared 14 days after pulse application.

Notes

  1. Liquid culture of BG-11 (containing 20 mM HEPES-KOH, pH 7.5 and 30 mM glucose) can be used (Fujita et al., 1992; Fujita et al., 1996, Fujita et al., 1998; Kimata-Ariga et al., 2000).
  2. To suspend cells on an agar plate, we use a sterile bent glass rod that is used for spreading Escherichia coli cells in transformation.
  3. Concentrated cell suspension may be too viscous to dispense by a normal micropipette tip. Thus, we prepare special tips (both of P-1000 and P-200 tips) by cutting the ends of normal tips with scissors (to be a diameter of approximately 2 mm) and used them for handling dense cell suspensions. These tips are autoclaved (121 °C, 20 min) before use.
  4. In the original procedure (Fujita et al., 1992), 1 mM HEPES-KOH (pH 7.5) and 10% (w/v) glycerol were sequentially used for washing cells. We recently found that using water instead gives essentially the same results as described here.
  5. Insufficient washing of cells and/or impurity of plasmid preparation may cause arc discharge in pulse application, resulting in transformation failure.
  6. Original settings were; voltage, 1.25 kV; capacitance 25 µF; and resistance, 600 ohms (Fujita et al., 1992). Using another electroporator (BTX Electro Cell Manipulator 600), the settings are; voltage, 1.45 kV; capacitance 50 µF; and resistance, 256 ohms.
  7. While only single recombinants may be isolated in one experiment, double recombinants can be eventually isolated in further trials (Figure 1). However, if double recombinants are never isolated even after many times trials of transformation, it is probable that the target gene is essential for growth under the conditions.
  8. Differentiation from a single recombinant to a double recombinant has never been observed even though many times of subculturing.
  9. For transformation with a shuttle vector, derivatives of pPBH201 (Kimata-Ariga et al., 2000; Yamamoto et al., 2009; Yamamoto et al., 2011), a relatively low concentration of plasmid (<0.1 µg µl-1) is enough to isolate transformants.

Recipes

  1. Ferric citrate solution
    60 mg citric acid
    60 mg ammonium iron (III) citrate, brown
    10 mg EDTA.Na2
  2. Trace metal A5+Co solution (1 L)
    2.86 g H3BO4
    1.81 g MnCl2.4H2O
    0.22 g ZnSO4.7H2O
    0.39 g Na2MoO4.2H2O
    79 mg CuSO4.5H2O
    49 mg Co(NO3)2.6H2O
  3. 2x BG-11 solution (1 L)
    3.0 g NaNO3 dissolved in 932 ml distilled water
    460 µl of 1 M K2HPO4 solution
    2.0 ml of 7.5% (w/v) MgSO4.7H2O
    2.0 ml of 3.6% (w/v) CaCl2.2H2O
    2.0 ml of 2.0% (w/v) Na2CO3
    20.0 ml of Ferric citrate solution
    Serially add 2.0 ml of Trace metal A5+Co
    40 ml of 1 M HEPES-KOH, pH 7.5
    Stored at 4 ºC
    Note: BG-11 liquid medium (Rippka et al., 1979) supplemented with 20 mM HEPES-KOH (pH 7.5). For BG-11 liquid medium, the equal amounts of 2x BG-11 and distilled water are mixed, and autoclaved (121 ºC, 20 min).
  4. BG-11 agar plates
    The equal amounts of 2x BG-11 and distilled water containing:
    Bacto Agar [3.0% (w/v)] is separately autoclaved (121 ºC, 20 min). Allow the media to cool about 50 ºC (Be careful not to be solidified at this stage). The solutions of 2x BG-11 and agar are mixed in a clean bench. If needed, appropriate antibiotic solution and glucose solution are added. Then, pour about 30 ml of medium into each Petri dish. When the BG-11 agar medium solidifies, invert the agar plates and store at 4 ºC.

Acknowledgments

This protocol was adapted from the previously published studies, Hiraide et al. (2015), Tsujimoto et al. (2014) and Fujita et al. (1992). The original protocol was described in Fujita et al. (1992), and we modified it as described here. We thank Yasuhiro Takahashi, Tomohiro Matsumura, Toshiharu Hase, and Hiroshi Matsubara for initial works on establishment of this transformation system. We thank Douglas K. Walton, Carl E. Bauer and Peter Wolk for donating plasmids pPBH201, pJRD215 and pRL425, respectively. We thank Chie Tomatsu for providing technical help. This work was supported by the Japan Society for the Promotion of Science (JSPS) (Grants-in-Aid for Scientific Research Nos. 05740481, 06740601, 07740617, 08836006, 11740445, 23370020, 23000007, 26660084, 15H04387 and 15H01397), Precursory Research for Embryonic Science and Technology (PRESTO) and the Advanced Low Carbon Technology Research and Development Program (ALCA).

References

  1. Davison, J., Heusterspreute, M., Chevalier, N., Ha-Thi, V. and Brunel, F. (1987). Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene 51(2-3): 275-280.
  2. Elhai, J. and Wolk, C. P. (1988). Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167: 747-754.
  3. Fujita, Y., Takagi, H. and Hase, T. (1996). Identification of the chlB gene and the gene product essential for the light-independent chlorophyll biosynthesis in the cyanobacterium Plectonema boryanum. Plant Cell Physiol 37(3): 313-323.
  4. Fujita, Y., Takagi, H. and Hase, T. (1998). Cloning of the gene encoding a protochlorophyllide reductase: the physiological significance of the co-existence of light-dependent and -independent protochlorophyllide reduction systems in the cyanobacterium Plectonema boryanum. Plant Cell Physiol 39(2): 177-185.
  5. Fujita, Y., Takahashi, Y., Chuganji, M. and Fujita, Y. (1992). The nifH-like (frxC) gene is involved in the biosynthesis of chlorophyll in the filamentous cyanobacterium Plectonema boryanum. Plant Cell Physiol 33(1): 81-92.
  6. Hiraide, Y., Oshima, K., Fujisawa, T., Uesaka, K., Hirose, Y., Tsujimoto, R., Yamamoto, H., Okamoto, S., Nakamura, Y., Terauchi, K., Omata, T., Ihara, K., Hattori, M. and Fujita, Y. (2015). Loss of cytochrome cM stimulates cyanobacterial heterotrophic growth in the dark. Plant Cell Physiol 56(2): 334-345.
  7. Kimata-Ariga, Y., Matsumura, T., Kada, S., Fujimoto, H., Fujita, Y., Endo, T., Mano, J., Sato, F. and Hase, T. (2000). Differential electron flow around photosystem I by two C(4)-photosynthetic-cell-specific ferredoxins. EMBO J 19(19): 5041-5050.
  8. Rao, R. N. and Rogers, S. G. (1979). Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments. Gene 7(1): 79-82.
  9. Rippka, Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Genetic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111, 1-61.
  10. Tsujimoto, R., Kamiya, N. and Fujita, Y. (2014). Transcriptional regulators ChlR and CnfR are essential for diazotrophic growth in nonheterocystous cyanobacteria. Proc Natl Acad Sci U S A 111(18): 6762-6767.
  11. Walton, D. K., Gendel, S. M. and Atherly, A. G. (1993). DNA sequence and shuttle vector construction of plasmid pGL3 from Plectonema boryanum PCC 6306. Nucleic Acids Res 21(3): 746.
  12. Yamamoto, H., Kurumiya, S., Ohashi, R. and Fujita, Y. (2009). Oxygen sensitivity of a nitrogenase-like protochlorophyllide reductase from the cyanobacterium Leptolyngbya boryana. Plant Cell Physiol 50(9): 1663-1673.
  13. Yamamoto, H., Kurumiya, S., Ohashi, R. and Fujita, Y. (2011). Functional evaluation of a nitrogenase-like protochlorophyllide reductase encoded by the chloroplast DNA of Physcomitrella patens in the cyanobacterium Leptolyngbya boryana. Plant Cell Physiol 52(11): 1983-1993.

简介

Leptolyngbya boryana ( L。boryana )(以前称为Plectonema boryanum)是一种多用途的丝状蓝细菌,具有在微氧条件下固氮的能力, 在黑暗中与葡萄糖异养生长,提供了研究光合作用,固氮和它们的调节机制的优良系统。 而 L。 boryana 不是天然可转化的,不同于单细胞蓝细菌集胞藻。 PCC 6803,它可以通过电穿孔转化。 这里我们描述了 L的变换。 boryana通过电穿孔分离其中靶基因被破坏的突变体。

关键字:蓝藻, 转型, 电穿孔, 靶向基因中断, 穿梭载体

材料和试剂

  1. 瓶顶过滤系统(0.22μm)(Corning,目录号:430624)
  2. 通过高压灭菌器(121℃,20分钟)灭菌的微量离心管(1.5ml)(Ina-optika corporation,BIO-BIK,目录号:ST-0150F)
  3. 微量移液器吸头(121oC,20分钟)
  4. 用于抗生素和葡萄糖溶液(Thermo Fisher Scientific,Millipore,目录号:SLGV033RV)或其等同物的过滤灭菌的无菌注射器过滤器(Millex-GV注射器过滤单元,0.22μm)
  5. 培养皿[无菌培养皿(90mm×15mm)](ASONE公司,目录号:1-7484-01-30)或其等同物
  6. 脉冲比色杯(Gene Pulser cuvette,0.1cm)(Bio-Rad Laboratories,AbD Serotec ,目录号:1652089)
  7. Hybond N +过滤器(圆盘直径82mm)(GE Healthcare,Amersham,目录号:RPN82B),通过高压灭菌器(121℃,20分钟)灭菌。
  8. (野生型或dg5 )(在补充有20mM HEPES-KOH,pH7.5和30mM葡萄糖的BG-11琼脂平板上生长) 注意:dg5菌株作为天然突变体从野生型分离,其在黑暗中比野生型生长快得多(Fujita等人,1996)。最近我们确定负责dg5表型的突变是一个腺嘌呤插入导致编码细胞色素c 的cytM基因中的移码(Hiraide等人,2015)。
  9. 灭菌水(150ml)
    注意:水通过RiOs Essential Water Purification System(Merck Millipore Corporation)或WEX系统(Yamato)纯化。也可以使用蒸馏水。高压灭菌(121℃,20分钟),使用前在冰上冷却。
  10. NaNO 3(Nacalai tesque,目录号:31617-35)
  11. 1 M K 2 HPO 4溶液(Wako Pure Chemical Industries,Siyaku,目录号:164-04295)
  12. 7.5%(w/v)MgSO 4 .7H 2 O(Nacalai tesque,目录号:21003-75)。
  13. 3.6%(w/v)CaCl 2 .0.2 2H 2 O(Nacalai tesque,目录号:06731-05)
  14. 2.0%(w/v)Na 2 CO 3(Nacalai tesque,目录号:31311-25)
  15. 柠檬酸(Wako Pure Chemical Industries,Siyaku,目录号:038-05521)
  16. 柠檬酸铁铵(III),棕色(Wako Pure Chemical Industries,Siyaku,目录号:092-00802)
  17. 乙二胺-N,N,N',N'-四乙酸二钠盐二水合物(EDTA-Na 2)(Dojindo,目录号:345-01865)
  18. H 3 BO 4(Wako Pure Chemical Industries,Siyaku,目录号:021-02195)
  19. (Nacalai tesque,目录号:21211-45)
  20. (Wako Pure Chemical Industries,Siyaku,目录号:268-00405)
  21. (Wako Pure Chemical Industries,Siyaku,目录号:197-02485)(日本化药工业株式会社,Siyaku,目录号:197-02485) )
  22. (Wako Pure Chemical Industries,Siyaku,目录号:033-04415)的紫外线吸收剂。
  23. (Wako Pure Chemical Industries,Siyaku,目录号:031 -03752)
  24. 1μM葡萄糖(过滤灭菌)(Wako Pure Chemical Industries,Siyaku,目录号:049-31165)
  25. 50mg/ml硫酸卡那霉素(过滤灭菌)(Wako Pure Chemical Industries,Siyaku,目录号:111-00344)
  26. 50mg/ml氯霉素(溶于乙醇)(Nacalai tesque,目录号:08027-72)
  27. 10mg/ml硫酸链霉素(过滤灭菌)(Meiji Seika Pharma,目录号:4987222665643)
  28. 50mg/ml红霉素(过滤灭菌)(Wako Pure Chemical Industries,Siyaku,目录号:054-05101)
  29. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(Nacalai tesque,目录号:17514-15)
  30. 质粒制备试剂盒(GenElute HP Plasmid Midiprep Kit)(Sigma-Aldrich,目录号:NA0200)
  31. 线性化质粒溶液(>2μgμl -1
    注意:在酶消化前,通过分光光度计测定质粒的浓度。
  32. 细菌琼脂[1.5%(w/v)](BD,目录号:214010)
  33. 柠檬酸铁溶液(见配方)
  34. 痕量金属A + Co溶液(1L)(参见配方)
  35. 2x BG-11溶液(1升)(见配方)
  36. 使用Bacto琼脂[1.5%(w/v)](BD,目录号:214010)固化的BG-11琼脂板(补充有20mM HEPES-KOH,pH 7.5,具有或不具有合适的抗生素,表1) )

设备

  1. 镊子
  2. 微量移液器(P-1000,P-200和P-20或其等同物)(Gilson)
  3. 清洁长椅
  4. 高压灭菌器(TOMY DIGITAL BIOLOGY CO。,型号:SX-500或其等同物)
  5. 基因脉冲Xcell(Bio-Rad Laboratories,AbD Serotec ,目录号:1652666,1652668和1652669)
  6. 分光光度计(Shimadzu,型号:UV-1700或GE Healthcare,型号:NanoVue Plus或其等同物)
  7. 吸气器(带真空泵)
  8. 生长柜(带适当的荧光灯泡)

程序

  1. 构建用于靶向诱变的质粒(图1)。质粒携带由靶基因的上游序列,药物抗性柱(表1)和靶基因的下游序列组成的嵌合DNA片段(图1B)。上游和下游序列应当各自大于1kb,用于稳定的同源重组。如果通过PCR获得上游和下游片段,则应当通过Sanger测序来确认其质粒中的核苷酸序列,以避免通过同源重组在靶基因外部引入非预期的突变。载体通常是pUC18/19衍生物。


    图1.质粒构建,同源重组和PCR中分析突变体的实例。 。A.在 L中靶基因( orf84 基因,LBWT_23230,以红色显示)的基因排列。 boryana (Tsujimoto ,,2014)。通过PCR扩增orf84基因的1.7kb上游和1.5kb下游片段(粗水平条)以构建质粒。 B.构建质粒pNK72以分离orf84阳性的突变体(Δorf84)。将两个扩增的片段克隆到载体中。在这种情况下,使用pUC192作为载体,其通过插入neo基因(来自pMC19,Fujita等人的卡那霉素抗性基因)构建。 ,1992)插入pUC19的BamHI位点(其中多克隆位点以黄色框显示的虚线)。将1.5-kb下游片段(用SalI-XhoI消化)克隆到pUC192的SalI位点,随后将1.7-kb上游片段(用BamHI消化)克隆到BamHI位点,得到质粒pNK72。 C.单个重组体的基因排列,其中质粒pNK72通过单个同源重组事件掺入染色体。野生型和敲除拷贝都存在于染色体中。在这种情况下,单重组事件发生在质粒的1.5-kb下游片段和相应的染色体部分之间。小的水平箭头表示用于PCR的引物,其显示在Δforf84突变体的基因排列中的基因排列中,其中orf84基因被替换为neo 基因通过双重重组事件。预先用KpnI和SalI消化质粒用于电穿孔以避免产生单个重组体。小的水平箭头表示在图F中显示的PCR引物。E.对orf84的单个重组体的PCR分析。检测到两个DNA片段。在三个转化体(泳道3-5)中,较长(实心三角形,1,799bp)和较短(开放三角形,437bp)片段分别对应于敲除和野生型拷贝的片段。在质粒pNK72作为模板的PCR中检测到较长的片段(泳道2)。在野生型细胞中检测到较短的片段(野生型拷贝)(泳道1)。 F.Δorf84的PCR分析。在两个转化体(泳道3和4)中只检测到对应于orf84的敲除拷贝的单个DNA片段(实心三角形,1,799bp)。较短和较长的片段(泳道1和2)与E相同(未公布的结果,Kotani和Fujita)。

  2. 由质粒制备试剂盒制备的质粒通过限制性酶线性化,以使质粒通过单重组事件整合到基因组中的可能性最小化,导致在二倍体中含有敲除和野生型拷贝的mer二倍体基因组(图1C)。消化位点可以选自载体部分中的任何位置。应该小心不要消化同源重组和药物抗性标记的部分。消化的质粒通过乙醇沉淀并溶解于无菌水中至>2μg/μl
  3. L。 boryana在含有30mM葡萄糖的BG-11的琼脂平板上在低光条件下(<10μmol s -1 )培养在30°C 2-7天(注1)。
  4. 以下所有程序应在无菌条件下进行。将琼脂平板上的细胞悬浮于无菌水(每个琼脂平板约2ml)中,并将悬浮液转移到瓶顶过滤器中(图2A-C。注释2和3)。在这个阶段,Hybond N +过滤器应该被放置在新的BG-11琼脂板上,用于在电穿孔后恢复细胞(步骤11)。
  5. 通过抽吸将细胞收集在瓶顶过滤器的表面上(图2D)

    图2.准备 L。 boryana 单元格用于脉冲应用

  6. 将在冰上冷却的约50ml的无菌水加入到过滤器中以悬浮细胞。通过抽吸再次将细胞收集在过滤器上。此洗涤程序重复三次(图3C。注释4-5)。离心(1,200×g,10分钟)是用水洗涤细胞的另一个选择。在这种情况下,请注意 L。由于长毛发,即使通过高速离心(例如,44,000×g/min,30分钟),boryana细胞形成非常松散的沉淀。
  7. 将过滤器上的细胞悬浮在小量等分试样(500μl)的无菌水中(图2D),并将悬浮液转移到微量离心管(1.5ml)中,在冰上储存直至脉冲施用(注3) >
  8. 将用于脉冲施用的悬浮液的小等分试样(50μl)分配到另一个微量离心管(注3)中,并将浓缩的质粒溶液(通常为10μl的2μgμl -1 )加入到管和混合好。
  9. 将混合物转移到间隙宽度为0.1cm的试管中并在冰上冷却(图2E-F,注释3)。
  10. 施加单指数衰减脉冲(设置:电压,1.41kV;电容,50μF;以及电阻,250欧姆)。时间常数约为10ms。时间常数和实际电压应记录在笔记上(注5-6)。
  11. 将BG-11液体培养基的等分试样(350μl)加入比色杯中,并用P-1000型微量移液器分配器的蓝色尖端回收细胞悬浮液(图3A),以铺展在覆盖在BG上的Hybond N + -11琼脂平板上(图3B-C)

    图3.从比色杯中恢复施加脉冲的细胞并在非选择性平板上接种

  12. 将琼脂平板在低光条件(<10μmolm-2 s -1 s -1 -1 )下在30℃下温育2天。在此期间,抗生素抗性基因被表达以赋予细胞对特定抗生素的抗性
  13. 将Hybond N +过滤器转移到含有合适抗生素的新BG-11琼脂板上(最终浓度显示在表1中)。将琼脂平板在30℃在高光条件下(约100μmolm -2 -2s -1 s -1)孵育。
  14. 显示对抗生素的抗性的菌落在约10至14天后出现(图4)。您可以用牙签或提示(对于P-200型微量移液管),应该预先高压灭菌(121°C,20分钟)。
  15. 一些菌落是单个重组体,一些是双重组体。应进行PCR分析以鉴定双重组体(图1E-F;注释7-8)
    表1.可用于转化L的抗生素列表。 boryana

    尚未用于定向诱变。然而,我们证实含有pJRD215(具有链霉素抗性基因)的转化体显示对链霉素的抗性(未公开的结果)。

代表数据

在靶基因破坏中的代表性结果显示在表2中。在大多数情况下,在选择性平板上出现的几乎所有转化体都是双重组体。重组接种转化体以分离其中所有野生型拷贝被突变体拷贝完全替换的细胞的程序在集胞藻属中是必需的。 PCC 6803,但是这个过程在 L中不需要。 boryana (注8)。

表2. L的电穿孔的实施例。 boryana

1
WT,野生型
2
脉冲施加的细胞悬浮液中的总DNA量
3
集落数。通过Southern印迹分析或PCR证实单或双重重组体
图4.转化体在选择性平板上的典型外观。在施用脉冲后14天出现两个微小的绿色菌落(红色箭头)。

笔记

  1. 可以使用BG-11(含有20mM HEPES-KOH,pH 7.5和30mM葡萄糖)的液体培养(Fujita等人,1992; Fujita em > ,1996,Fujita等人,1998; Kimata-Ariga等人,2000)。 br />
  2. 为了将细胞悬浮在琼脂平板上,我们使用用于在转化中扩散大肠杆菌细胞的无菌弯曲玻璃棒。
  3. 浓缩的细胞悬浮液可能太粘稠,不能通过正常的微量移液管吸头分配。因此,我们通过用剪刀(直径约2mm)切割常规尖端的末端并用于处理致密的细胞悬浮液,来制备特殊的尖端(P-1000和P-200尖端)。这些提示在使用前进行高压灭菌(121℃,20分钟)
  4. 在原始程序(Fujita等人,1992)中,依次使用1mM HEPES-KOH(pH7.5)和10%(w/v)甘油洗涤细胞。我们最近发现,使用水代替基本上与此处所述的结果相同。
  5. 如果细胞和/或质粒制备物的杂质不足,可能会在脉冲应用中导致电弧放电,导致转化失败
  6. 原始设置是;电压,1.25kV;电容25μF;和电阻,600欧姆(Fujita等人,1992)。使用另一个电穿孔仪(BTX Electro Cell Manipulator 600),设置为:电压,1.45kV;电容50μF;和电阻,256欧姆
  7. 尽管在一个实验中可以仅分离单个重组体,但是可以在进一步的试验中最终分离双重组体(图1)。然而,如果即使在多次转化试验之后双重组体也从未分离,则靶基因可能在该条件下对于生长是必需的。
  8. 从单个重组体到双重组体的分化从未被观察到,即使进行许多次传代培养。
  9. 对于用穿梭载体的转化,pPBH201的衍生物(Kimata-Ariga等人,2000; Yamamoto等人,2009; Yamamoto等人, 2011),相对低浓度的质粒(<0.1μgμl -1 )足以分离转化体。

食谱

  1. 柠檬酸铁溶液
    60mg柠檬酸
    60mg柠檬酸铁铵(III),棕色
    10毫克EDTA。 Na
  2. 痕量金属A 5+ Co溶液(1L)
    2.86g H sub 3 BO sub 4
    1.81g MnCl 2 4H 2 O 0.22g ZnSO 4 7H O 0.39g Na 2 SO 4 MoO 4 sub。 2H 2 O 79mg CuSO 4 。 5H O
    49mg Co(NO 3)sub 2+。 6H 2 O
  3. 2x BG-11溶液(1L)
    3.0g NaNO 3溶解在932ml蒸馏水中 460μl的1M K 2 HPO 4溶液
    2.0ml的7.5%(w/v)MgSO 4 .7H 2 O 2。 2.0ml的3.6%(w/v)的CaCl 2 .2H 2 O 2。 2.0ml的2.0%(w/v)Na 2 CO 3 3 / 20.0ml柠檬酸铁溶液 连续加入2.0ml痕量金属A + Co
    40ml 1M HEPES-KOH,pH7.5 储存在4oC
    注:补充有20mM HEPES-KOH(pH 7.5)的BG-11液体培养基(Rippka等人,1979)。对于BG-11液体培养基,将等量的2×BG-11和蒸馏水混合,并高压灭菌(121℃,20分钟)。
  4. BG-11琼脂平板上 等量的2x BG-11和蒸馏水,含有:
    细菌琼脂[3.0%(w/v)]分别高压灭菌(121℃,20分钟)。让介质冷却约50oC(小心不要在这个阶段固化)。将2x BG-11和琼脂的溶液在清洁工作台中混合。如果需要,加入适当的抗生素溶液和葡萄糖溶液。然后,向每个培养皿中倒入约30ml培养基。当BG-11琼脂培养基凝固时,颠倒琼脂平板并在4℃保存

致谢

该方案改编自以前发表的研究,Hiraide等(2015),Tsujimoto等人(2014)和Fujita 等人(1992)。原始方案在Fujita等人(1992)中描述,并且我们如本文所述对其进行修改。我们感谢高桥康夫,松村俊,Toshiharu Hase和松原浩先生关于建立这个转换系统的初步工作。我们感谢道格拉斯K.沃尔顿,卡尔E.鲍尔和彼得沃尔克分别捐赠质粒pPBH201,pJRD215和pRL425。我们感谢Chie Tomatsu提供技术帮助。这项工作得到日本科学促进会(JSPS)(科学研究助理科学研究编号05740481,06740601,07740617,08836006,11740445,2370020,23000007,2666084,15H04387和15H01397)的支持,Precursory Research胚胎科学技术(PRESTO)和先进的低碳技术研究与开发计划(ALCA)。

参考文献

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  3. Fujita,Y.,Takagi,H。和Hase,T。(1996)。鉴定在蓝细菌中的光独立的叶绿素生物合成所必需的chlB 基因和基因产物。 植物细胞生理学 37(3):313-323
  4. Fujita,Y.,Takagi,H。和Hase,T。(1998)。 克隆编码原叶绿素还原酶的基因:光依赖性共存的生理学意义和植物细胞生理学39(2):177-185。植物细胞生理学39(2):177-185。
  5. Fujita,Y.,Takahashi,Y.,Chuganji,M.and Fujita,Y。(1992)。 nifH 样( frxC )基因参与丝状蓝细菌藻类的叶绿素的生物合成。


    植物细胞生理学33(1):81-92。 >
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引用:Tsujimoto, R., Kotani, H., Nonaka, A., Miyahara, Y., Hiraide, Y. and Fujita, Y. (2015). Transformation of the Cyanobacterium Leptolyngbya boryana by Electroporation. Bio-protocol 5(24): e1690. DOI: 10.21769/BioProtoc.1690.
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