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
发布: 2016年07月05日第6卷第13期 DOI: 10.21769/BioProtoc.1855 浏览次数: 8035
评审: Maria SinetovaChristian RothDamián Lobato-Márquez
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参见作者原研究论文
The authors used this protocol in:
Sep 2015
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
- Plastic disposable dish (for BG-11 plates)
- 1.5 ml microtubes
- Synechococcus elongatus PCC 7942
- Escherichia coli K-12 W3110
- Chloramphenicol (Nacalai tesque, catalog number: 06285 )
- Glutaraldehyde solution (50% in water) (KANTO KAGAKU, catalog number: 17026-32 )
- Tween 20 (KANTO KAGAKU, catalog number: 40350-02 )
- Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D5652 )
- Liquid nitrogen
- Trisodium citrate dihydrate (KANTO KAGAKU, catalog number: 37150-00 )
- SYTOX Green Nucleic Acid Stain (5 mM solution in DMSO) (Thermo Fisher Scientific, catalog number: S7020 )
- BG-11 liquid medium (Castenholz, 1988) (see Recipes)
- BG-11 plates (see Recipes)
- 50x BG-11 stock (see Recipes)
- 1,000x Micro elements A6 (see Recipes)
- 1,000x K2HPO4 (see Recipes)
- 1,000x CaCl2 (see Recipes)
- 1,000x Ammonium Iron(lll) citrate brown (see Recipes)
- 1,000x Na2S2O3 (see Recipes)
- 1 M HEPES-KOH (pH 8.2) (see Recipes)
- M9 medium (Sambrook et al., 1989) (see Recipes)
- 10x M9 salt (see Recipes)
- PBS buffer (see Recipes)
Equipment
- Plant growth chamber (TOMY, model: CLE-405 )
- Growth chamber (YAMATO, model: IC602 )
- Laboratory shaker (TAITEC, model: PERSONAL11 )
- UV/Vis spectrophotometer (Shimadzu, model: UV-1800 )
- Pharmaceutical refrigerator (Panasonic, model: MPR-3120CN-PJ )
- Rotator (TAITEC, model: RT-50 )
- FACS Calibur instrument (BD Biosciences, FACSCaliburTM)
- 100 ml test tubes (for culturing)
- Microtube centrifuge (TOMY, model: MX-107 )
Software
- Analytical software: BD CellQuest Pro (Becton-Dickinson)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- 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.
- 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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分类
分子生物学 > DNA > DNA 定量
微生物学 > 微生物遗传学 > DNA
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