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Estimation of the Chromosomal Copy Number in Synechococcus elongatus PCC 7942
聚球藻PCC 7942中染色体拷贝数的估测   

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Abstract

Cyanobacteria are prokaryotic organisms that perform oxygenic photosynthesis. Freshwater cyanobacteria, such as Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, are model organisms for the study of photosynthesis, gene regulation, and biotechnological applications because they are easy to manipulate genetically. However, while studying these cyanobacteria, care has to be taken with respect to genetic heterogeneity in the establishment of gene disruptants, because these cyanobacteria contain multiple chromosomal copies per cell. Here, we describe a method for the estimation of chromosomal copy number in Synechococcus 7942. Using this method, we have recently observed that the chromosomal copy number of Synechococcus 7942 significantly changes during its growth phases. This technique is available for studying polyploidy not only in cyanobacteria, but also in other polyploid organisms.

Keywords: Cyanobacteria(蓝藻), Ploidy(倍性), Bacterial Cell Cycle(细菌的细胞周期)

Materials and Reagents

  1. Plastic disposable dish (for BG-11 plates)
  2. 1.5 ml microtubes
  3. Synechococcus elongatus PCC 7942
  4. Escherichia coli K-12 W3110
  5. Chloramphenicol (Nacalai tesque, catalog number: 06285 )
  6. Glutaraldehyde solution (50% in water) (KANTO KAGAKU, catalog number: 17026-32 )
  7. Tween 20 (KANTO KAGAKU, catalog number: 40350-02 )
  8. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D5652 )
  9. Liquid nitrogen
  10. Trisodium citrate dihydrate (KANTO KAGAKU, catalog number: 37150-00 )
  11. SYTOX Green Nucleic Acid Stain (5 mM solution in DMSO) (Thermo Fisher Scientific, catalog number: S7020 )
  12. BG-11 liquid medium (Castenholz, 1988) (see Recipes)
  13. BG-11 plates (see Recipes)
  14. 50x BG-11 stock (see Recipes)
  15. 1,000x Micro elements A6 (see Recipes)
  16. 1,000x K2HPO4 (see Recipes)
  17. 1,000x CaCl2 (see Recipes)
  18. 1,000x Ammonium Iron(lll) citrate brown (see Recipes)
  19. 1,000x Na2S2O3 (see Recipes)
  20. 1 M HEPES-KOH (pH 8.2) (see Recipes)
  21. M9 medium (Sambrook et al., 1989) (see Recipes)
  22. 10x M9 salt (see Recipes)
  23. PBS buffer (see Recipes)

Equipment

  1. Plant growth chamber (TOMY, model: CLE-405 )
  2. Growth chamber (YAMATO, model: IC602 )
  3. Laboratory shaker (TAITEC, model: PERSONAL11 )
  4. UV/Vis spectrophotometer (Shimadzu, model: UV-1800 )
  5. Pharmaceutical refrigerator (Panasonic, model: MPR-3120CN-PJ )
  6. Rotator (TAITEC, model: RT-50 )
  7. FACS Calibur instrument (BD Biosciences, FACSCaliburTM)
  8. 100 ml test tubes (for culturing)
  9. Microtube centrifuge (TOMY, model: MX-107 )

Software

  1. Analytical software: BD CellQuest Pro (Becton-Dickinson)

Procedure

  1. Preparation of Synechococcus 7942 culture
    1. Synechococcus 7942 is streaked and pre-incubated on BG-11 agar plates at 30 °C under continuous illumination (40 µE/m2/s) for 1 week in the plant growth chamber.
    2. Cells from the plates are suspended in approximately 1 ml of BG-11 medium with high turbidity and diluted into 80 ml of BG-11 medium at an OD750 of 0.05.
    3. The culture is incubated at 30 °C under continuous illumination (40 µE/m2/s) with 2% CO2 bubbling for 10 days.
    4. The culture is then diluted to OD750 = 0.2 with fresh BG-11 medium. After 18-h incubation under dark conditions at 30 °C with 2% CO2 bubbling, the culture is transferred to light conditions (40 µE/m2/s) to restart cell growth.
    5. A 1-ml aliquot of the cultured cells is used for the following assay (Procedure C, D and E).

  2. Preparation of E. coli culture as a standard
    1. E. coli is streaked and pre-incubated on M9 agar plates at 37 °C in the growth chamber.
    2. Cells from the plates are suspended in approximately 1 ml of M9 medium with high turbidity and then diluted into 10 ml of M9 medium at an OD600 of 0.2.
    3. The culture is incubated at 30 °C with shaking for 4 h.
    4. 10 µl of 20 mg/ml chloramphenicol is added to the culture (final concentration, 20 µg/ml).
    5. After incubation with shaking for 2 h, 1 ml of cell culture is subjected to the following assay.

  3. Fixation
    1. To the 1-ml aliquots of cell culture, 10 µl of 0.5% Tween 20 and 20 µl of 50% glutaraldehyde are added (final conc. 0.005% and 1%, respectively), and incubated for 30 min at 4 °C with gentle agitation by a rotator.
    2. The cells are centrifuged at 20,000 x g for 1 min and then washed with 1 ml of PBS, and the cell pellets were stored at -30 °C until further analysis.

  4. DNA staining
    1. Cell pellets were frozen with liquid N2, and then thawed at room temperature.
      Note: This step is necessary for the permeation of SYTOX Green.
    2. The cell pellets are resuspended in 50 µl of 10 µM SYTOX Green solution, which was diluted with 50 mM trisodium citrate (pH 8.0).
    3. The samples are incubated overnight at 4 °C.

  5. FACS analysis
    1. The SYTOX Green-stained cells are subjected to FACS analysis. The stained cell suspension (5 µl) is diluted with 250 µl of PBS and immediately analyzed by FACS.
    2. The cells could be selected by cell size and internal complexity by using forward (FSC) and side scatter (SSC) values. The SYTOX Green signal corresponds to the DNA amount, and is detected by the FL1 detector (laser: 488 nm, filter: 533/30 nm) (Figures 1 and 2).
      Note: The signal of Synechococcus 7942 cells can be identified based on chlorophyll autofluorescence using FL3 (laser: 488 nm, filter: 610/20 nm).
    3. The major peak of the chloramphenicol-treated E. coli sample, which corresponds to 1 copy of the chromosome per E. coli cell, is extracted and used as a standard for estimating the chromosomal copy number.

  6. Estimation of chromosomal copy number
    The Synechococcus 7942 chromosome copy number (ca. 2.7 Mbp) is calculated based on the peak location of E. coli (ca. 4.7 Mbp) (Figure 3). The copy number of Synechococcus 7942 changes under our culture conditions (2-4 copies under dark and 3-8 copies under light conditions) (Watanabe et al. 2015) as reported previously (Binder and Chisholm, 1995; Griese et al., 2011).
    Equation: Copy number of the Synechococcus 7942 chromosome = FL1 valueSynechococcus x 4.7 (Genome size in E. coli)/FL1 valueE.coli x 2.7 (Genome size in Synechococcus

Representative data


Figure 1. DNA content profiles of chloramphenicol-treated E. coli cells. After culturing for 2 h in M9 medium with 20 µg/ml chloramphenicol, the cells are fixed and analyzed. A. Profiles of cell size (X axis: FSC-H) and DNA content (Y axis). B. Profiles of DNA content (X axis) and cell number (Y axis). For preparing the standard peak, which corresponds to single copy chromosome in E. coli, the major peak (asterisk) was extracted. The bottom panel represents the profiles obtained after extraction by gating.


Figure 2. The DNA content profiles of cells cultured in the dark (Top) and lag-phase Synechococcus 7942 cells (bottom) cultured in light conditions for 9 h. A. Profiles of cell size (X axis: FSC-H) and DNA content (Y axis). B. Profiles of DNA content (X axis) and cell number (Y axis).


Figure 3. DNA content profiles of cells cultured in the dark (thin line), lag-phase Synechococcus 7942 cells cultured in light conditions for 9 h (bold line), and the chloramphenicol-treated E. coli culture (thin red line). The copy number of the Synechococcus 7942 chromosome (ca. 2.7 Mbp) was calculated based on the peak location of E. coli (ca. 4.7 Mbp).

Recipes

  1. BG-11 liquid medium (pH 8.2)/1 L
    50x BG-11 stock 20 ml
    1,000x Micro elements A6 1 ml
    1,000x K2HPO4 1 ml
    1,000x CaCl2 1 ml
    1,000x Ammonium Iron(lll) citrate brown 1 ml
    1 M HEPES-KOH (pH 8.2) 20 ml
    H2O up to 1 L
    Autoclave (A. C.) (120 °C, 20 min)
  2. BG-11 plates (pH 8.2)/1 L
    1. 2x BG-11 stock
      50x BG-11 stock 20 ml
      1,000x Micro elements A6 1 ml
      1,000x K2HPO4 1 ml
      1,000x CaCl2 1 ml
      1,000x Ammonium Iron(lll) citrate brown 1 ml
      1,000x Na2S2O3 1 ml
      1 M HEPES-KOH (pH 8.2) 20 ml
      H2O up to 500 ml
      A. C. (120 °C, 20 min)
    2. 2x Agar stock
      Bacto agar (Nissui) 15 g
      H2O 500 ml
      A. C. (120 °C, 20 min)
      After dissolve Recipe 2b, mix with Recipe 2a.
  3. 50x BG-11 stock, 1 L
    NaNO3 75 g
    MgSO4·7H2O 3.75 g
    Citric acid 0.33 g
    EDTA-3Na Salt 0.05 g
    Na2CO3 1.0 g
    H2O up to 1 L
    A. C. (120 °C, 20 min)
  4. 1,000x Micro elements A6, 1 L
    H3BO3 2.86 g
    MnCl2·4H2O 1.81 g
    ZnSO4·7H2O 0.222 g
    Na2MoO4·2H2O 0.391 g
    Co(NO3)2·6H2O 0.0494 g
    CuSO4·5H2O 0.08 g
    H2O up to 1 L
    A. C. (120 °C, 20 min)
  5. 1,000x K2HPO4, 100 ml
    K2HPO4 3.0 g
    H2O up to 100 ml
    A. C. (120 °C, 20 min)
  6. 1,000x CaCl2, 100 ml
    CaCl2·2H2O 3.6 g
    H2O up to 100 ml
    A. C. (120 °C, 20 min)
  7. 1,000x Ammonium Iron(lll) citrate brown, 100 ml
    Ammonium Iron(lll) citrate brown 0.6 g
    H2O up to 100 ml
    A. C. (120 °C, 20 min)
  8. 1,000x Na2S2O3, 100 ml
    Na2S2O3 15.81 g
    H2O up to 100 ml
    A. C. (120 °C, 20 min)
  9. 1 M HEPES-KOH (pH 8.2), 1 L
    HEPES 238.3 g
    pH adjusted to 8.2 with KOH
    A. C. (120 °C, 20 min)
  10. M9 medium, 1 L
    H2O (A. C.) 895 ml
    10x M9 salt 100 ml
    1 M MgSO4 (A. C.) 1 ml
    40% Glucose (A. C., 120 °C, 15 min) 5 ml
    1% Thiamine-HCl (A. C.) 1 ml
    1M CaCl2 (A. C.) 100 µl
  11. 10x M9 salt, 1 L
    Na2HPO4 60 g
    KH2PO4 30 g
    NaCl 5 g
    NH4Cl 10 g
    H2O up to 1 L
    A. C. (120 °C, 20 min)
  12. PBS buffer
    PBS powder 9.6 g
    H2O up to 1 L
    A. C. (120 °C, 20 min)

Acknowledgments

This protocol was adapted from Watanabe et al. (2012). This work was supported in part by a Grants-in-Aid 25850056 from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S. W.

References

  1. Binder, B. J. and Chisholm, S. W. (1995). Cell cycle regulation in marine Synechococcus sp. strains. Appl Environ Microbiol 61(2): 708-717.
  2. Castenholz, R. W. (1988). Culturing methods for cyanobacteria. Meth Enzymol 167: 68-93.
  3. Griese, M., Lange, C. and Soppa, J. (2011). Ploidy in cyanobacteria. FEMS Microbiol Lett 323(2): 124-131.
  4. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Bacterial media, antibiotics, and bacterial strains. In: Molecular cloning. A laboratory manual. Cold Spring Harbor, A.1-13.
  5. Watanabe, S., Ohbayashi, R., Shiwa, Y., Noda, A., Kanesaki, Y., Chibazakura, T. and Yoshikawa, H. (2012). Light-dependent and asynchronous replication of cyanobacterial multi-copy chromosomes.Mol Microbiol 83(4): 856-865.
  6. Watanabe, S., Ohbayashi, R., Kanesaki, Y., Saito, N., Chibazakura, T., Soga, T. and Yoshikawa, H. (2015). Intensive DNA replication and metabolism during the lag phase in cyanobacteria. PLoS One 10(9): e0136800.

简介

蓝细菌是进行含氧光合作用的原核生物。 淡水蓝细菌,例如细长聚球藻PCC 7942和聚集细胞 PCC 6803是用于研究光合作用,基因调节和生物技术应用的模式生物,因为它们易于基因操作。 然而,当研究这些蓝细菌时,必须注意遗传异质性在基因破坏物的建立中,因为这些蓝细菌每个细胞含有多个染色体拷贝。 在这里,我们描述了用于估计在聚球藻7942中的染色体拷贝数的方法。使用这种方法,我们最近观察到,聚球藻7942的染色体拷贝数显着地改变 其生长阶段。 这种技术可用于研究多倍体不仅在蓝细菌,而且在其他多倍体生物。

关键字:蓝藻, 倍性, 细菌的细胞周期

材料和试剂

  1. 塑料一次性盘(BG-11板)
  2. 1.5 ml微管
  3. 细长聚球藻 PCC 7942
  4. 大肠杆菌 K-12 W3110
  5. 氯霉素(Nacalai tesque,目录号:06285)
  6. 戊二醛溶液(50%水溶液)(KANTO KAGAKU,目录号:17026-32)
  7. Tween 20(KANTO KAGAKU,目录号:40350-02)
  8. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D5652)
  9. 液氮
  10. 柠檬酸三钠二水合物(KANTO KAGAKU,目录号:37150-00)
  11. SYTOX Green核酸染料(5mM DMSO溶液)(Thermo Fisher Scientific,目录号:S7020)
  12. BG-11液体培养基(Castenholz,1988)(参见Recipes)
  13. BG-11板(见配方)
  14. 50x BG-11股票(见配方)
  15. 1,000x微元件A6(参见配方)
  16. (参见配方)

  17. 1,000x CaCl <2> (见配方)
  18. 1,000x柠檬酸铁铵(III)棕色(见配方)
  19. (参见配方)
  20. 1 M HEPES-KOH(pH 8.2)(参见配方)
  21. M9培养基(Sambrook等人,1989)(参见Recipes)
  22. 10x M9盐(参见配方)
  23. PBS缓冲液(见配方)

设备

  1. 植物生长室(TOMY,型号:CLE-405)
  2. 生长室(YAMATO,型号:IC602)
  3. 实验室振动器(TAITEC,型号:PERSONAL11)
  4. UV/Vis分光光度计(Shimadzu,型号:UV-1800)
  5. 药用冰箱(Panasonic,型号:MPR-3120CN-PJ)
  6. Rotator(TAITEC,型号:RT-50)
  7. FACS Calibur仪器(BD Biosciences,FACSCalibur TM
  8. 100ml试管(用于培养)
  9. Microtube离心机(TOMY,型号:MX-107)

软件

  1. 分析软件:BD CellQuest Pro(Becton-Dickinson)

程序

  1. 聚球藻7942培养物的制备
    1. 将聚球藻7942在30℃在连续照明(40μE/m 2/s)下在BG-11琼脂平板上划线并预孵育1周,在植物中生长室
    2. 将来自板的细胞悬浮于约1ml具有高浊度的BG-11培养基中,并稀释到OD为750的PBS为80的BG-11培养基中。
    3. 将培养物在30℃下在具有2%CO 2鼓泡的连续照明(40μE/m 2/s)下温育10天。
    4. 然后用新鲜的BG-11培养基将培养物稀释至OD 750 = 0.2。在黑暗条件下在30℃,2%CO 2鼓泡下孵育18小时后,将培养物转移至光条件(40μE/m 2 s/s)至重新启动细胞生长
    5. 将1ml培养细胞的等分试样用于以下测定(程序C,D和E)
  2. E的制备。大肠杆菌培养物作为标准
    1. E。将大肠杆菌在生长室中在37℃下在M9琼脂平板上划线并预孵育。
    2. 将来自板的细胞悬浮于约1ml具有高浊度的M9培养基中,然后稀释到OD 600为0.2的M9培养基中。
    3. 将培养物在30℃下振荡孵育4小时
    4. 向培养物中加入10μl20mg/ml氯霉素(终浓度20μg/ml)。
    5. 在振荡温育2小时后,将1ml细胞培养物进行以下测定
  3. 固定
    1. 向1ml等分的细胞培养物中加入10μl0.5%Tween 20和20μl50%戊二醛(最终浓度分别为0.005%和1%),并在4℃下温和温育30分钟通过旋转器搅拌
    2. 将细胞以20,000×g离心1分钟,然后用1ml PBS洗涤,将细胞沉淀储存在-30℃直到进一步分析。

  4. DNA染色
    1. 将细胞沉淀用液N 2 2冷冻,然后在室温下解冻。
      注意:此步骤对于SYTOX Green
      的渗透是必要的
    2. 将细胞沉淀重悬于50μl10μMSYTOX Green溶液中,用50mM柠檬酸三钠(pH8.0)稀释。
    3. 样品在4℃下孵育过夜。

  5. FACS分析
    1. 将SYTOX Green染色的细胞进行FACS分析。将染色的细胞悬浮液(5μl)用250μlPBS稀释,并立即通过FACS分析。
    2. 通过使用正向(FSC)和侧向散射(SSC)值,可以通过细胞大小和内部复杂性来选择细胞。 SYTOX Green信号对应于DNA量,并且通过FL1检测器(激光:488nm,过滤器:533/30nm)检测(图1和图2)。
      注意:可以使用FL3 em(激光:488nm,滤光片:610/20nm)基于叶绿素自发荧光来鉴定聚球藻7942细胞的信号。
    3. 氯霉素处理的主峰。大肠杆菌样品,其对应于每个E的染色体的1个拷贝。大肠杆菌细胞,被提取并用作估计染色体拷贝数的标准
  6. 染色体拷贝数的估计
    基于em的峰位置计算 Synechococcus 7942染色体拷贝数(约2.7Mbp)。大肠杆菌(约4.7Mbp)(图3)。在我们的培养条件下(黑暗下2-4个拷贝,光照条件下3-8个拷贝)改变了聚球蓝细菌7942的拷贝数(Watanabe等人,2015) (Binder和Chisholm,1995; Griese等人,2011)。
    方程式:聚球藻7942染色体的拷贝数= FL1值聚球藻 ×4.7(大肠杆菌中的基因组大小 )/FL1值大肠杆菌 ×2.7(聚球藻中的基因组大小)</b>

代表数据


图1.氯霉素处理的DNA含量曲线。 。在含有20μg/ml氯霉素的M9培养基中培养2小时后,固定并分析细胞。 A.细胞大小(X轴:FSC-H)和DNA含量(Y轴)的分布。 B.DNA含量(X轴)和细胞数(Y轴)的谱。用于制备标准峰,其对应于E中的单拷贝染色体。大肠杆菌,提取主峰(星号)。底面板表示通过门控提取后获得的轮廓

图2.在光照条件下培养9小时的在黑暗(上)和滞后期聚球藻7942细胞(底部)中培养的细胞的DNA含量概况。细胞大小(X轴:FSC-H)和DNA含量(Y轴)的分布。 B.DNA含量(X轴)和细胞数(Y轴)的轮廓

图3.在光照条件下培养9小时(黑体)的黑暗(细线),滞后期聚球藻7942细胞中培养的细胞的DNA含量曲线。 strong> ,以及氯霉素处理的。大肠杆菌培养物(红色细线)。基于 E的峰位置计算集胞藻 7942染色体(约2.7Mbp)的拷贝数。大肠杆菌(约4.7 Mbp)。

食谱

  1. BG-11液体培养基(pH8.2)/1L
    50x BG-11储液20ml,
    1,000x微元件A6 1 ml
    1,000×K <2> HPO <4> 1ml
    1,000x CaCl 2 2ml/dm2 1,000x柠檬酸铁棕(lll)1 ml
    1 M HEPES-KOH(pH 8.2)20ml
    H sub 2 O最多1 L
    高压灭菌(A.C.)(120℃,20分钟)
  2. BG-11板(pH 8.2)/1L
    1. 2x BG-11股票
      50x BG-11储液20ml,
      1,000x微元件A6 1 ml
      1,000×K <2> HPO <4> 1ml
      1,000x CaCl 2 2ml/dm2 1,000x柠檬酸铁棕(lll)1 ml
      1,000×Na <2> 2 O 1 ml
      1 M HEPES-KOH(pH 8.2)20ml
      H sub 2 O最多500 ml
      (120℃,20分钟)
    2. 2x琼脂原液
      Bacto琼脂(Nissui)15g
      H sub 2 O 500ml
      (120℃,20分钟) 溶解食谱2b后,与食谱2a混合
  3. 50x BG-11股票,1 L
    NaNO <3> 75g
    MgSO 4·7H 2 O 3.75g/g 柠檬酸0.33克
    EDTA-3Na盐0.05g
    Na sub 2 CO 3 sub 1.0g
    H sub 2 O最多1 L
    (120℃,20分钟)
  4. 1,000x微元件A6,1 L

    BO 3 2.86 g MnCl 2 2·4H 2 O 1.81g
    ZnSO 4·7H 2 O 0.222g
    Na 2 2 MoO 4 4·2H 2 O 0.391g
    (NO 3)2 O 6·6H 2 O 0.0494g
    CuSO 4·5H 2 O 0.08g/g H sub 2 O最多1 L
    (120℃,20分钟)
  5. 1,000×K HPO <4>,100ml
    HPO 3.0g
    H <2> O高达100毫升
    (120℃,20分钟)
  6. 1,000x CaCl 2 2,100ml
    CaCl 2·2H 2 O 3.6g·
    H <2> O高达100毫升
    (120℃,20分钟)
  7. 1,000x柠檬酸铁铵(III)棕色,100ml
    柠檬酸铁铵(III)褐色0.6g
    H <2> O高达100毫升
    (120℃,20分钟)
  8. 1000×Na <2> 2 3 ,100ml

    O高达100毫升
    (120℃,20分钟)
  9. 1 M HEPES-KOH(pH 8.2),1L
    HEPES 238.3克
    用KOH调节pH至8.2 (120℃,20分钟)
  10. M9培养基,1 L
    H 2 O(A.C.)895ml
    10x M9盐100ml
    1M MgSO 4(A.C.)1ml
    40%葡萄糖(A.C.,120℃,15分钟)5ml
    1%硫胺-HCl(A.C.)1ml
    1M CaCl 2(A.C.)100μl
  11. 10x M9盐,1L

    HPO 60
    KH 2 PO 4 30 g
    NaCl 5 g/h NH 4 Cl 10 g/g H sub 2 O最多1 L
    (120℃,20分钟)
  12. PBS缓冲区
    PBS粉末9.6g
    H sub 2 O最多1 L
    (120℃,20分钟)

致谢

该协议改编自Watanabe等人(2012)。这项工作得到了日本教育,文化,体育,科学和技术部给S.W.的赠款25850056的部分支持。

参考文献

  1. Binder,BJ和Chisholm,SW(1995)。  Cell在海洋聚球藻中的循环调节。菌株。 Appl Environ Microbiol 61(2):708-717。
  2. Castenholz,RW(1988)。  蓝细菌的培养方法。 Meth Enzymol 167:68-93
  3. Griese,M.,Lange,C.和Soppa,J.(2011)。  Ploidy in cyanobacteria。 FEMS Microbiol Lett 323(2):124-131。
  4. Sambrook,J.,Fritsch,E.F.and Maniatis,T。(1989)。细菌培养基,抗生素和细菌菌株。在:分子克隆。 A laboratory manual。 Cold Spring Harbor,A.1-13。
  5. Watanabe,S.,Ohbayashi,R.,Shiwa,Y.,Noda,A.,Kanesaki,Y.,Chibazakura,T.and Yoshikawa,H。 蓝藻多拷贝染色体的光依赖和异步复制。/a> Mol Microbiol 83(4):856-865。
  6. Watanabe,S.,Ohbayashi,R.,Kanesaki,Y.,Saito,N.,Chibazakura,T.,Soga,T.和Yoshikawa,H。(2015)。  在蓝藻中的滞后期内强烈的DNA复制和代谢。 PLoS One 10(9):e0136800。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Watanabe, S. and Yoshikawa, H. (2016). Estimation of the Chromosomal Copy Number in Synechococcus elongatus PCC 7942. Bio-protocol 6(13): e1855. DOI: 10.21769/BioProtoc.1855.
  2. Watanabe, S., Ohbayashi, R., Kanesaki, Y., Saito, N., Chibazakura, T., Soga, T. and Yoshikawa, H. (2015). Intensive DNA replication and metabolism during the lag phase in cyanobacteria. PLoS One 10(9): e0136800.
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