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Stable Transformation of Cyanobacterium Synechocystis sp.
集胞藻细胞的稳定转化   

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Abstract

Cyanobacteria are prokaryotes, which perform oxygenic photosynthesis. Among them, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a well characterized model system for studies on oxygenic photosynthesis, light signal transduction etc. Moreover, Synechocystis is applied in biotechnological applications (Desai and Atsumi, 2013). Stable transformation of Synechocytis is achieved via the uptake of DNA and incorporation into the host genome by homologous double recombination. This allows for the generation of gene knock-outs (KO) by replacing the coding sequence of the gene of interest by a KO-cassette (comprising of a selection marker flaked by sequences of the gene of interest) or stable overexpression of certain genes of interest after insertion of a corresponding overexpression cassette at a neutral insertion site on the host genome. Stable transformation of Synechocystis was reported by Grigorieva and Shestakov (1982). Since then, variants of the initial protocol have been applied successfully to transform Synechocystis sp. Here we describe a lab-protocol that was applied successfully for stable transformation of Synechocystis (Schwarzkopf et al., 2014).

Keywords: Transformation(转型), Cyanobacteria(蓝藻), Synechocystis sp.(集胞藻)

Materials and Reagents

  1. Synechocystis sp. PCC 6803 wild-type (WT) strain [see Schwarzkopf et al. (2014) for details]
  2. Antibiotics
    1. Chloramphenicol (Merck KGaA, catalog number: 2366 )
    2. Kanamycin sulfat (Carl Roth, catalog number: T832.1 )
    3. Spectinomycin (Duchefa Biochemie, catalog number: S 0188.0025 )
  3. Phyto agar (Duchefa Biochemie, catalog number: P1003.1000 )
  4. NaNO3 (Carl Roth, catalog number: 8601.2 )
  5. K2HPO4 (Carl Roth, catalog number: P749.2 )
  6. MgSO4.7 H2O (Carl Roth, catalog number: P027.2 )
  7. CaCl2.2 H2O (Sigma-Aldrich, catalog number: 223506 )
  8. Citric acid (Carl Roth, catalog number: 1818.1 )
  9. Ferric ammonium citrate (III+) (Carl Roth, catalog number: CN77.1 )
  10. EDTA Na2 (Carl Roth, catalog number: 8043.2 )
  11. Na2CO3 (Sigma-Aldrich, catalog number: S-1641 )
  12. H3BO3 (Carl Roth, catalog number: 6943.3 )
  13. MnCl2.4 H2O (Sigma-Aldrich, catalog number: M3634 )
  14. ZnSO4.7 H2O (Merck KGaA, catalog number: 0 143532 )
  15. Na MoO4.5 H2O (Carl Roth, catalog number: 0274.3 )
  16. CuSO4.5 H2O (Carl Roth, catalog number: P025.1 )
  17. Co(NO3)2.6 H2O (Merck KGaA, catalog number: A834336548 )
  18. Na2S2O3.5 H2O (Merck KGaA, catalog number: K5023616 )
  19. BG11 medium (see Recipes)
  20. BG11 agar plates (see Recipes)
  21. Common antibiotics used (see Recipes)

Equipment

  1. Petri dishes (Greiner Bio-one, catalog number: 632180 )
  2. 2 ml-reaction tubes (Eppendorf)
  3. Tape (Gotha-VLIES, 10 m x 1.25 cm) (Gothaplast, catalog number: PZN-7105417 )
  4. Centrifuge (Eppendorf, model: 5810R )
  5. Shaking incubator with illumination (Sartorius, model: Certomat® BS-T )
  6. Light shelves provided with light bulbs (NARVA LT 36W/760-010 daylight) (Brand-Erbisdorf)
  7. Photometer (Pharmacia LKB-Ultrospec III)
  8. Flow cabinet (Heraeus, HERAsafe, model: HS12 )

Procedure

  1. All the handling of Synechocystis cells was done in a flow cabinet. Sterile working conditions are critical throughout the whole procedure.
  2. Synechocystis cells were maintained on BG11 agar plates under continuous illumination (20 μmol/m2/s) at 28 °C and were restreaked in 2-week intervals.
  3. To generate batch cultures, 25 ml liquid BG11 medium (in a 100-ml-glass beaker) was inoculated with one inoculation loop of Synechocystis cells and cells were grown to an optical density (OD750) of 0.5 to 0.8 (mid-exponential growth) under continuous illumination at a light intensity of 20 μmol/m2/s at 28 °C and permanent agitation (150 rpm). Depending on the inoculation density it takes several days to about one week to reach the indicated OD750 of 0.5 to 0.8.
  4. 10 ml of cell cultures were centrifuged (5 min, 28 °C, 1,107 x g) and the cell pellet was resuspended in 5 ml BG11 medium and equally distributed to five sterile 2 ml-reaction tubes.
  5. DNA (3 to 5 μg of circular plasmid DNA) was added and cells were gently mixed by tapping and incubated in darkness at 28 °C over night. To measure the viability and spontaneous mutation frequencies one aliquot was left without plasmid DNA and incubated in darkness at 28 °C over night.
  6. Following dark incubation, 200 μl of the cell cultures were plated on BG11 agar plates (94 x 16 mm, see “Materials” for details) lacking antibiotics and incubated in continuous light at a light intensity of 20 μmol/m2/s at 28 °C.
  7. After 2 days of incubation under constant light, the agar plates were supplied with antibiotics at a concentration of 15 μg/ml. Therefore, the agar was lifted with a sterile spatula and 1 ml of the appropriate antibiotic solution was applied to the bottom of the petri dish (Figure 1). In case several different antibiotics have to be applied prepare an antibiotic mixture containing each antibiotic combined in 1 ml of total volume.
  8. Plates were sealed with tape to avoid drying-out and further incubated under continuous illumination at a light intensity of 20 μmol/m2/s at 28 °C. Colonies appeared after 2 to 3 weeks.
  9. To achieve complete segregation, single colonies were picked and streaked on BG11 plates containing the appropriate antibiotics (see step 10 for appropriate antibiotic concentrations). When cells were gown (about one to two weeks) cells were streaked again on new BG11 plates containing the appropriate antibiotics. This successive streak purification was repeated at least four rounds (in total) to ensure segregation.
  10. To check for positive genomic integration of the construct PCR on genomic DNA was performed. It might happen that complete segregation cannot be achieved even after several rounds of successive streak purification. This might happen when a gene knock-out is attempted of a gene, which is indispensable for viability of the cells. In such a case anti-sense approaches or overexpression of dominant negative forms might be applied to interfere with the function of the gene of interest.
  11. Positive clones were maintained on BG11 agar plates containing appropriate antibiotics under continuous illumination (20 μmol/m2/s) and were restreaked in 2-week intervals. Common antibiotics used are chloramphenicol at 8 μg/ml, spectinomycin at 10 μg/ml, kanamycin at 10 μg/ml.


    Figure 1. Applying antibiotics beneath the agar layer. A sterile curved spatula was used to lift the agar layer and antibiotic solutions were applied to the bottom of the petri dish.

Notes

  1. Transformation efficiency may vary, depending on the cell status and the construct used for transformation and commonly result in 10 to 50 clones per approach.

Recipes

  1. BG11 medium (Stanier et al., 1971)
    BG11 medium was prepared in 10x concentration, autoclaved at 121 °C for 12 min and stored at 4 °C. Before use, 890 ml H2Odest, 100 ml 10x BG11 and 10 ml of the trace metal solution were combined to yield 1L of 1x BG11. BG11 medium was aliquoted in culture vessels, autoclaved and stored at 4 °C.
    10x BG11 medium:
    NaNO3 15 g
    K2HPO4 0.4 g
    MgSO4.7 H2O 0.75 g
    CaCl2.2 H2O 0.36 g
    Citric acid 0.06 g
    Ferric ammonium citrate (III+) 0.06 g
    EDTA Na2 0.01 g
    Na2CO3 0.2 g
    Add H2Odest 1 L
    Autoclaved and stored at 4 °C
    Trace metal solution per litre:
    H3BO3 2.86 g
    MnCl2.4 H2O 1.81 g
    ZnSO4.7 H2O 0.22 g
    Na MoO4.5 H2O 0.39 g
    CuSO4.5 H2O 0.08 g
    Co(NO3)2.6 H2O 0.05 g
  2. BG11 agar plates
    For solid medium 1x BG11 was supplemented with 1.5 % (w/v) phyto agar, 0.18 % (w/v) sodium thiosulfate (Na2S2O3.5 H2O; added for better solidification of the agar), autoclaved and poured in petri-dishes (approximately 30 ml per dish). Where appropriate, filter-sterilized antibiotics were added to the medium.
  3. Common antibiotics used
    Chloramphenicol at 8 μg/ml
    Spectinomycin at 10 μg/ml
    Kanamycin at 10 μg/ml

Acknowledgments

Stable transformation of Synechocystis was reported by Grigorieva and Shestakov (1982). Experimental work was supported by Technische Universität München (TUM) core funding.

References

  1. Desai, S. H. and Atsumi, S. (2013). Photosynthetic approaches to chemical biotechnology. Curr Opin Biotechnol 24(6): 1031-1036.
  2. Grigorieva, G. and Shestakov, S. (1982). Transformation in the cyanobacterium Synechocystis sp. 6803. FEMS Microbiol Lett 13(4): 367-370.
  3. Schwarzkopf, M., Yoo, Y. C., Huckelhoven, R., Park, Y. M. and Proels, R. K. (2014). Cyanobacterial phytochrome2 regulates the heterotrophic metabolism and has a function in the heat and high-light stress response. Plant Physiol 164(4): 2157-2166.
  4. Stanier, R. Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35(2): 171-205.

简介

蓝细菌是原核生物,其执行含氧光合作用。其中,单细胞蓝细菌集胞藻 PCC 6803(下文称为"集胞藻")是用于研究含氧光合作用,光信号转导等的良好表征的模型系统。此外,集胞藻应用于生物技术应用(Desai和Atsumi,2013)。通过DNA的吸收和通过同源双重重组并入宿主基因组中实现集胞藻的稳定转化。这允许通过用KO盒(包含由感兴趣的基因的序列片段化的选择标记)或感兴趣的基因的某些基因的稳定过表达来产生基因敲除(KO)在相应的过表达盒插入宿主基因组上的中性插入位点之后。稳定转化集胞藻由Grigorieva和Shestakov(1982)报道。从那时起,初始协议的变体已经成功应用于变换集胞藻。在这里,我们描述了成功应用于集胞藻的稳定转化的实验室方案(Schwarzkopf等人,2014)。

关键字:转型, 蓝藻, 集胞藻

材料和试剂

  1. 集胞藻 PCC 6803野生型(WT)菌株[参见Schwarzkopf et al。(2014)了解详情]
  2. 抗生素
    1. 氯霉素(Merck KGaA,目录号:2366)
    2. 卡那霉素硫酸盐(Carl Roth,目录号:T832.1)
    3. 壮观霉素(Duchefa Biochemie,目录号:S 0188.0025)
  3. 植物琼脂(Duchefa Biochemie,目录号:P1003.1000)
  4. NaNO 3(Carl Roth,目录号:8601.2)

  5. HPO 4(Carl Roth,目录号:P749.2)
  6. MgSO 4·7H 2 O(Carl Roth,目录号:P027.2)
  7. CaCl 2 2(Sigma-Aldrich,目录号:223506)。
  8. 柠檬酸(Carl Roth,目录号:1818.1)
  9. 柠檬酸铁铵(III )(Carl Roth,目录号:CN77.1)
  10. EDTA Na 2(Carl Roth,目录号:8043.2)
  11. Na 2 CO 3(Sigma-Aldrich,目录号:S-1641)

  12. (Carl Roth,目录号:6943.3)
  13. (Sigma-Aldrich,目录号:M3634)。
  14. ZnSO 4·7H 2 O(Merck KGaA,目录号:0143532)

  15. 5(Carl Roth,目录号:0274.3)
  16. (Carl Roth,目录号:P025.1)
  17. (Merck KGaA,目录号:A834336548),br(NO 3)2 sub。 />
  18. (Merck KGaA,Ltd。),其包含一种或多种选自以下的化合物:Na 2 O 3,Na 2 O 3, 目录号:K5023616)
  19. BG11介质(见配方)
  20. BG11琼脂平板(见配方)
  21. 使用的常见抗生素(参见配方)

设备

  1. 培养皿(Greiner Bio-one,目录号:632180)
  2. 2ml反应管(Eppendorf)
  3. 胶带(Gotha-VLIES,10m×1.25cm)(Gothaplast,目录号:PZN-7105417)
  4. 离心机(Eppendorf,型号:5810R)
  5. 使用照明摇动培养箱(Sartorius,型号:Certomat BS-T)
  6. 带有灯泡的轻型货架(NARVA LT 36W/760-010日光)(Brand-Erbisdorf)
  7. 光度计(Pharmacia LKB-Ultrospec III)
  8. 流动柜(Heraeus,HERAsafe,型号:HS12)

程序

  1. 所有对集胞藻细胞的处理在流动箱中进行。 在整个过程中,无菌工作条件至关重要。
  2. 将集胞藻细胞在28℃下在连续照射(20μmol/m 2 s/s)下维持在BG11琼脂板上,并以2周的间隔重新划线。
  3. 为了产生分批培养物,用一个接种环的集胞藻细胞接种25ml液体BG11培养基(在100ml-玻璃烧杯中),并使细胞生长至光密度(OD 750在28℃下在20μmol/m 2的光强度的连续照明下的中等指数生长(中指数生长)和持续的搅拌(150rpm)。取决于接种密度,需要几天至约一周以达到0.5至0.8的指示OD 750值。
  4. 将10ml细胞培养物离心(5分钟,28℃,1×10 7×g),将细胞沉淀重悬于5ml BG11培养基中,并均匀分布于5个无菌的2ml反应管中。
  5. 加入DNA(3至5μg环状质粒DNA),通过轻拍轻轻混合细胞,并在黑暗中在28℃下温育过夜。为了测量存活率和自发突变频率,留下一个等分试样,没有质粒DNA,并在黑暗中在28℃下温育过夜。
  6. 在黑暗孵育后,将200μl细胞培养物接种在缺乏抗生素的BG11琼脂平板(94×16mm,参见"材料")上,并在连续光中以20μmol//sup>/s
  7. 在恒定光照下培养2天后,向琼脂平板中加入浓度为15μg/ml的抗生素。因此,用无菌刮刀提起琼脂,并将1ml适当的抗生素溶液施加到培养皿的底部(图1)。在必须应用几种不同的抗生素的情况下,制备含有在1ml总体积中组合的每种抗生素的抗生素混合物。
  8. 用胶带密封板,以避免干燥,并在28℃下在20μmol/m 2的光强度的连续照明下进一步温育。 2至3周后出现菌落。
  9. 为了实现完全分离,挑取单个菌落,并在含有合适抗生素的BG11平板上划线(参见步骤10,获得合适的抗生素浓度)。当细胞长出(约一至两周)时,将细胞再次在含有合适抗生素的新BG11平板上划线。将该连续条纹纯化重复至少四轮(总共)以确保分离。
  10. 为了检查构建体的阳性基因组整合,对基因组DNA进行PCR。可能发生即使在几轮连续条纹纯化后也不能实现完全分离。当尝试基因敲除基因时,这可能发生,该基因对于细胞的活力是不可缺少的。在这种情况下,可以应用反义方法或显性负面形式的过表达以干扰目标基因的功能。
  11. 在连续照射(20μmol/m 2/s/s )下,将阳性克隆保持在含有适当抗生素的BG11琼脂平板上,并以2周的间隔重新划线。使用常用抗生素 是8μg/ml的氯霉素,10μg/ml的壮观霉素,10μg/ml的卡那霉素。


    图1.在琼脂层下施加抗生素。使用无菌弯曲刮刀提起琼脂层,并将抗生素溶液施加到培养皿的底部。

笔记

  1. 转化效率可以变化,这取决于细胞状态和用于转化的构建体,并且通常每种方法导致10至50个克隆。

食谱

  1. BG11培养基(Stanman等人,1971)
    BG11培养基以10x浓度制备,在121℃高压灭菌12分钟并储存在4℃。在使用前,将890ml H 2 Odest,100ml 10×BG11和10ml痕量金属溶液合并,得到1L 1×BG11。将BG11培养基等分到培养容器中,高压灭菌并在4℃贮存 10x BG11介质:
    NaNO <3> 15g
    HP 0.4g
    MgSO 4·7H 2 O 7·7H 2 O 0.75·g / CaCl 2 2 H sub 2 O 0.36 g
    柠檬酸0.06 g
    柠檬酸铁铵(III +)0.06g
    EDTA Na 2 0.01g/g Na 2 CO 3 sub 3 0.2g
    添加H 2 Odest 1 L
    高压灭菌并在4°C下贮存
    每升痕量金属溶液:

    MnCl 2 4 H 2 O 1.81g
    ZnSO 4 sub
    7 H sub 2 O 0.22 g
    Na MoO 4 H 0.5 H 2 O 0.39g
    CuSO 4 H 5 H 2 O 0.08g/dm 2 Co(NO 3)2.6 H 2 O 0.05g/m 2
  2. BG11琼脂平板上 对于固体培养基1,BG11补充有1.5%(w/v)植物琼脂,0.18%(w/v)硫代硫酸钠(Na 2 S 2 Sub 2 O) 加入以更好地凝固琼脂),高压灭菌并倒入陪替氏培养皿(每个培养皿约30ml)中。 适当时,将过滤灭菌的抗生素加入培养基中
  3. 使用常用抗生素
    氯霉素8μg/ml
    10μg/ml的壮观霉素
    卡那霉素10μg/ml

致谢

稳定转化集胞藻由Grigorieva和Shestakov(1982)报道。实验工作由慕尼黑理工大学(TUM)核心资助。

参考文献

  1. Desai,S. H.和Atsumi,S。(2013)。 光合作用的化学生物技术 Opin Biotechnol 24 (6):1031-1036。
  2. Grigorieva,G。和Shestakov,S。(1982)。 转换在蓝细菌集胞藻 FEMS Microbiol Lett 13(4):367-370。
  3. Schwarzkopf,M.,Yoo,Y. C.,Huckelhoven,R.,Park,Y.M.and Proels,R.K。(2014)。 蓝藻植物色素2调节异养代谢,并在热和高光应激反应中具有功能。/a> Plant Physiol 164(4):2157-2166。
  4. Stanier,R.Y.,Kunisawa,R.,Mandel,M。和Cohen-Bazire,G。(1971)。 单细胞蓝绿藻的纯化和性质 细菌学杂志35(2):171-205。
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Copyright: © 2014 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. Proels, R. K. (2014). Stable Transformation of Cyanobacterium Synechocystis sp.. Bio-protocol 4(21): e1286. DOI: 10.21769/BioProtoc.1286.
  2. Schwarzkopf, M., Yoo, Y. C., Huckelhoven, R., Park, Y. M. and Proels, R. K. (2014). Cyanobacterial phytochrome2 regulates the heterotrophic metabolism and has a function in the heat and high-light stress response. Plant Physiol 164(4): 2157-2166.
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