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Burkholderia glumae Competent Cells Preparation and Transformation
水稻细菌性谷枯病菌感受态细胞的制备和转化   

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Abstract

Bukholderia glumae is a gram-negative bacterium which causes grain rot, seedling rot and panicle blight in rice and bacterial wilt in many field crops. This bacterium has been reported from major rice growing regions around the world and is now considered as an emerging major pathogen of rice (Tsushima et al., 1996; Jeong et al., 2003; Kim et al., 2010; Ham et al., 2011). Here we describe two methods for competent cells preparation and transformation of B. glumae. Using these methods, we have applied effector detector system (Sohn et al., 2007) to B. glumae (Sharma et al., 2013).

Keywords: Burkholderia glumae(水稻细菌性谷枯病菌), Competent cells(感受态细胞), Transformation(转型), PEDV5 based vectors(pedv5载体)

Materials and Reagents

  1. Burkholderia glumae strain 106619 (National Institute of Agricultural Sciences Genebank, Tsukuba, Ibaraki, Japan)
  2. pEDV5 based vectors (Fabro et al., 2011; Sharma et al., 2013)
  3. Gentamycin
  4. Tryptone
  5. Yeast extract
  6. Protease peptone no. 3
  7. Ice
  8. Lysogeny Broth (LB) medium (see Recipes)
  9. King’s Broth (KB) agar (see Recipes)
  10. KB agar with 25 ng/ml gentamycin (see Recipes)
  11. 10% glycerol (see Recipes)
  12. 300 mM sucrose solution (see Recipes)

Equipment

  1. Deep freezer
  2. Autoclave
  3. Clean bench
  4. Petri plates
  5. Sterile 1.5 ml tubes
  6. Sterile 15 ml tubes
  7. Sterile 50 ml tubes
  8. Incubation Shaker
  9. Aluminum foil
  10. Spectrophotometer
  11. Centrifuge
  12. Toothpick
  13. MicroPulser/Gene Pulser Cuvettes, 0.2 cm gap (Bio-Rad Laboratories, catalog number: 165-2089 )
  14. MicroPulser/Gene Pulser Cuvettes, 0.1 cm gap (Bio-Rad Laboratories, catalog number: 165-2086 )
  15. Gene Pulser XcellTM Electroporation Systems (Bio-Rad Laboratories)
  16. Minisart filters (pore size 0.2 μm) (Sigma-Aldrich, catalog number: 16534K )
  17. Disposable Cell Spreaders

Procedure

 Part I: Conventional method

  1. Preparation of competent cells
    1. 10 μl of glycerol stock of the B. glumae strain is inoculated to 20 ml of LB medium in a 50 ml tube and further incubated at 28 °C for 16-40 h with horizontal shaking at 200 rpm until OD600 = 0.8 is achieved.
    2. The lid of the tube is opened for 30 sec under a clean bench.
    3. The tube is incubated again at 28 °C for 4 h with horizontal shaking at 200 rpm.
    4. The tube is centrifuged twice at 800 x g at 4 °C for 5 min.
    5. Each time the pellet is dissolved in 20 ml of autoclaved cold 10% glycerol.
    6. The pellet is dissolved in 200 μl of cold 10% glycerol and divided into 50 μl aliquots and stored at -80 °C for a further transformation step.
  2. Transformation
    1. Remove from -80 °C tubes containing 50 μl of electro-competent B. glumae cells.
    2. Thaw the cells on ice.
    3. Add ~1 μg of pEDV5 based vector into the B. glumae cells. Incubate on ice up to 3 min.
    4. Transfer the mixture of cells + DNA to a cold electroporation cuvette (0.2 cm electrode gap). Make sure the suspension is at the bottom of the cuvette.
    5. Set the Gene Pulser apparatus at 25 μF the volt at 2.5 kV. Set the Pulse resistance controller to 200 ohms.
    6. Place the cold cuvette in the chamber slide (Cuvette notch facing away from you).
    7. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
    8. Electroporate by pushing the red button.
    9. Remove the cuvette from the chamber and immediately add 1 ml of LB medium to the cuvette and quickly resuspend the cells by pipetting.
    10. Transfer the cell suspension from the cuvette to 1.5 ml tubes and incubate on shaker (200 rpm) at 28 °C for 2 h to allow recovery and expression of the gentamycin resistance marker (Clean cuvettes successively with dH2O, EtOH, sterile water, then wrap with aluminum foil then autoclave).
    11. Pipette 200 μl of each transformation on KB agar plates containing 25 ng/ml gentamycin.
    12. Spread the cells with cell spreaders. Place plates inverted at 28 °C for 2-3 days in the dark.

 Part II. High competency method

  1. Preparation of competent cells
    1. A frozen glycerol stock of the B. glumae strain is picked with a toothpick and spread on a KB agar plate.Place plates inverted at 28 °C for 2-3 days in the dark.
    2. Freshly grown B. glumae colony is inoculated to 5 ml of LB medium in a 15 ml tube and further incubated at 28 °C for 16 h with horizontal shaking at 200 rpm.
    3. 1 ml aliquots is centrifuged twice at 3,500 x g at 4 °C for 5 min.
    4. Each time the pellet is dissolved in 1 ml of filter sterilized and room temperature 300 mM sucrose solution.
    5. The pellet is dissolved in 200 μl of 300 mM sucrose solution and divided into 100 μl aliquots and used the cells immediately for a further transformation step.
  2. Transformation
    1. Add ~1 μg of pEDV5 based vector into the B. glumae cells.
    2. Transfer the mixture of cells + DNA to an electroporation cuvette (0.1 cm electrode gap). Make sure the suspension is at the bottom of the cuvette.
    3. Set the Gene Pulser apparatus at 25 μF the volt at 1.8 kV. Set the Pulse resistance controller to 200 ohms.
    4. Place the cuvette in the chamber slide (Cuvette notch facing away from you).
    5. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
    6. Electroporate by pushing the red button.
    7. Remove the cuvette from the chamber and immediately add 1 ml of LB medium to the cuvette and quickly resuspend the cells by pipetting.
    8. Transfer the cell suspension from the cuvette to 1.5 ml tubes and incubate on shaker (200 rpm) at 28 °C for 2 h to allow recovery and expression of the gentamycin resistance marker (Clean cuvettes successively with dH2O, EtOH, sterile water, then wrap with aluminum foil then autoclave).
    9. Pipette 200 μl of each transformation on KB agar plates containing 25 ng/ml gentamycin.
    10. Spread the cells with cell spreaders. Place plates inverted at 28 °C for 2-3 days in the dark.

Recipes

  1. LB medium
    Mix 5 g of tryptone
    2.5 g of yeast extract
    5 g of NaCl with 800 ml dH2O
    Add 0.2 ml of 5 N NaOH
    Add dH2O to 1,000 ml and autoclave it for 20 min
  2. KB agar
    Mix 20 g of protease peptone no. 3
    10 g of glycerol
    1.5 g of K2HPO4
    1.5 g of MgSO4.7H2O with 800 ml dH2O
    Adjust to pH 7.2
    Add dH2O to 1,000 ml, add 15 g of agar and autoclave it for 20 min
  3. KB agar with 25 ng/ml gentamycin
    Cool down the autoclaved KB agar to 50 °C
    Add 1 ml of 25 mg/ml gentamycin
    Pour in 90 mm x 15 mm Perti dish
  4. 10% glycerol
    Mix 100 g of glycerol with 900 ml dH2O and autoclave it for 20 min
  5. 300 mM sucrose solution
    Mix 10.27 g of sucrose with 80 ml
    Add dH2O to 100 ml and filter (0.2 μm) sterilize it

Acknowledgments

This protocol was adapted from Sharma et al. (2013). This work was supported by the ‘Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN)’ (Japan) and Japan Society for the Promotion of Science (JSPS) grants no. 18310136 and 20380027, and ‘The Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation PMI-0010)’ and the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research on Innovative Areas 23113009); and by JSPS grant nos 22780040 and 2301518 to H. Saitoh, JSPS grant no. 2200214 to R. Terauchi. The financial assistance received from JSPS to Shailendra Sharma and Shiveta Sharma for carrying out this study is gratefully acknowledged. S. Kamoun and J.D.G. Jones were supported by The Gatsby Foundation (United Kingdom).

References

  1. Ham, J. H., Melanson, R. A. and Rush, M. C. (2011). Burkholderia glumae: next major pathogen of rice? Mol Plant Pathol 12(4): 329-339.
  2. Jeong, Y., Kim, J., Kim, S., Kang, Y., Nagamatsu, T. and Hwang, I. (2003). Toxoflavin produced by Burkholderia glumae causing rice grain rot is responsible for inducing bacterial wilt in many field crops. Plant Disease 87(8): 890-895.
  3. Kim, J., Kang, Y., Kim, J.G., Choi, O. and Hwang, I. (2010). Occurrence of Burlkholderia glumae on rice and field crops in Korea. Plant Pathol J 26(3): 271-272.
  4. Sharma, S., Sharma, S., Hirabuchi, A., Yoshida, K., Fujisaki, K., Ito, A., Uemura, A., Terauchi, R., Kamoun, S., Sohn, K. H., Jones, J. D. and Saitoh, H. (2013). Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells. Plant J 74(4): 701-712. 
  5. Sohn, K. H., Lei, R., Nemri, A. and Jones, J. D. (2007). The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana. Plant Cell Online 19(12): 4077-4090.
  6. Tsushima, S., Naito, H. and Koitabashi, M. (1996). Population dynamics of Pseudomonas glumae, the causal agent of bacterial grain rot of rice, on leaf sheaths of rice plants in relation to disease development in the field. Ann Phytopathol Soci Japan 62(2): 108-113.

简介

Bukholderia glumae 是一种革兰氏阴性细菌,其在水稻中引起谷物腐烂,幼苗腐烂和穗病,在许多田间作物中引起细菌性枯萎。 这种细菌已经从世界上的主要水稻生长区域报道,并且现在被认为是水稻的新出现的主要病原体(Tsushima等人,1996; Jeong等人, ,2003; Kim ,2010; Ham ,,2011)。 在这里我们描述了感受态细胞制备和转化的两种方法。 glumae 。 使用这些方法,我们已经将效应检测器系统(Sohn等人,2007)应用于B。 glumae (Sharma ,,2013)。

关键字:水稻细菌性谷枯病菌, 感受态细胞, 转型, pedv5载体

材料和试剂

  1. 菌株106619(National Institute of Agricultural Sciences Genebank,Tsukuba,Ibaraki,Japan)
  2. 基于pEDV5的载体(Fabro等人,2011; Sharma等人,2013)。
  3. 庆大霉素
  4. 胰蛋白酶
  5. 酵母提取物
  6. 蛋白酶蛋白胨 3

  7. 溶菌酶肉汤(LB)培养基(参见食谱)
  8. 王氏肉汤(KB)琼脂(见配方)
  9. KB琼脂与25ng/ml庆大霉素(参见Recipes)
  10. 10%甘油(参见配方)
  11. 300 mM蔗糖溶液(见配方)

设备

  1. 深冷冻机
  2. 高压灭菌器
  3. 清洁长椅
  4. 培养皿
  5. 无菌1.5 ml试管
  6. 无菌15 ml试管
  7. 无菌50ml管
  8. 孵育振荡器
  9. 铝箔
  10. 分光光度计
  11. 离心机
  12. 牙签
  13. MicroPulser/Gene Pulser比色杯,0.2cm间隙(Bio-Rad Laboratories,目录号:165-2089)
  14. MicroPulser/Gene Pulser比色杯,0.1cm间隙(Bio-Rad Laboratories,目录号:165-2086)
  15. Gene Pulser Xcell TM 电穿孔系统(Bio-Rad Laboratories)
  16. Minisart过滤器(孔径0.2μm)(Sigma-Aldrich,目录号:16534K)
  17. 一次性电池分布器

程序

  第I部分:传统方法

  1. 感受态细胞的制备
    1. 10μl甘油 将荚壳炭疽杆菌株接种到20ml的LB培养基中 50ml管中,并在28℃下进一步用水平温育16-40小时 在200rpm振摇直到OD 600 = 0.8。
    2. 在洁净台上将管的盖打开30秒。
    3. 将管在28℃下再次温育4小时,在200rpm下水平摇动。
    4. 将管在4℃下以800×g离心两次5分钟。
    5. 每次将沉淀溶解在20ml高压灭菌的冷10%甘油中。
    6. 的   将沉淀溶于200μl冷的10%甘油中并分成50μl μl等分试样并储存在-80℃用于进一步的转化步骤。
  2. 转型
    1. 从-80℃的管中取出含有50μl电感受态的B。 glumae 细胞。
    2. 解冻冰上的细胞。
    3. 加入约1μg的基于pEDV5的载体到。 glumae 细胞。 在冰上孵育至3分钟。
    4. 转让   将细胞+ DNA的混合物加入冷的电穿孔小杯(0.2cm 电极间隙)。 确保悬浮液在底部 比色皿。
    5. 将Gene Pulser装置设置为25μF,电压为2.5 kV。 将脉冲电阻控制器设置为200欧姆。
    6. 将冷的比色皿放在室滑道(比色杯凹口面向远离你)。
    7. 将玻片推入样品室,直到样品池位于样品室底部的触点之间。
    8. 通过按红色按钮电击。
    9. 去掉   来自室的比色皿并立即加入1ml LB培养基 并通过吸移快速重悬细胞。
    10. 转让   将细胞悬浮液从试管中取出至1.5ml管中并孵育 振荡器(200rpm)在28℃下2小时以允许恢复和表达 庆大霉素抗性标记(依次用dH 2 O 2清洁试管,   EtOH,无菌水,然后用铝箔包裹,然后高压灭菌)。
    11. 吸取200μl的每个转化在含有25ng/ml庆大霉素的KB琼脂平板上。
    12. 用细胞扩散器扩散细胞。 将板在28℃下在黑暗中颠倒2-3天

  第二部分。 高能力方法

  1. 感受态细胞的制备
    1. A的冷冻甘油原液。 用牙签挑取腮腺炎菌株并铺在KB琼脂平板上。平板在28℃下在黑暗中翻转2-3天。
    2. 新鲜的   生长B. 葫芦科菌落接种到15ml LB培养基中的5ml LB培养基中 并在28℃下水平振荡孵育16小时 200rpm。
    3. 将1ml等分试样在4℃以3,500×g离心两次5分钟。
    4. 每次将沉淀溶解于1ml过滤灭菌的和室温的300mM蔗糖溶液中。
    5. 的   将沉淀溶于200μl300mM蔗糖溶液中并分配 到100μl等分试样,并立即使用细胞进一步 变换步骤。
  2. 转型
    1. 将〜1μg基于pEDV5的载体添加到荚壳芽孢杆菌细胞中。
    2. 转让   细胞+ DNA的混合物到电穿孔小杯(0.1cm 电极间隙)。 确保悬浮液在底部 比色皿。
    3. 将Gene Pulser装置设置为25μF,电压为1.8 kV。 将脉冲电阻控制器设置为200欧姆。
    4. 将试管放置在腔室载玻片(比色杯凹口背向您)。
    5. 将玻片推入样品室,直到样品池位于样品室底部的触点之间。
    6. 通过按红色按钮电击。
    7. 去掉   来自室的比色皿并立即加入1ml LB培养基 并通过吸移快速重悬细胞。
    8. 转移细胞悬浮液从试管到1.5毫升管和 在振荡器(200rpm)上在28℃孵育2小时以允许恢复和 表达庆大霉素抗性标记(Clean cuvettes 依次用dH 2 O,EtOH,无菌水,然后用铝箔包裹,然后高压灭菌)。
    9. 吸取200μl的每个转化在含有25ng/ml庆大霉素的KB琼脂平板上。
    10. 用细胞扩散器扩散细胞。 将板在28℃下在黑暗中颠倒2-3天

食谱

  1. LB培养基
    混合5 g胰蛋白酶
    2.5g酵母提取物
    5g NaCl和800ml dH 2 O
    加入0.2ml 5N NaOH
    将dH 2 O加入到1,000ml中并高压灭菌20分钟
  2. KB琼脂
    将20g蛋白胨蛋白胨 3
    10g甘油 1.5g K 2 HPO 4
    1.5g的MgSO 4·7H 2 O与800ml dH 2 O·
    调至pH 7.2
    将dH 2 O加入到1,000ml中,加入15g琼脂,并高压灭菌20分钟。
  3. KB琼脂和25ng/ml庆大霉素 将高压灭菌的KB琼脂冷却至50℃
    加入1ml 25mg/ml庆大霉素
    倒入90 mm x 15 mm Perti盘
  4. 10%甘油 将100g甘油与900ml dH 2 O混合,并将其高压灭菌20分钟
  5. 300mM蔗糖溶液 将10.27g蔗糖与80ml
    混合 将dH 2 O加到100ml中,并过滤(0.2μm)使其消毒

致谢

该方案改编自Sharma等人(2013)。这项工作得到了"促进创新生物科学基础研究活动计划(PROBRAIN)"(日本)和日本科学促进会(JSPS)赠款号的支持。 18310136和20380027,以及"日本农业,林业和渔业部(农业创新基因组学PMI-0010)"和日本教育,文化,体育,科学和技术部(科学技术研究所创新区域研究23113009);和JSPS授权号22780040和2301518授予H.Saitoh,JSPS授权号。 2200214到R.Terauchi。非常感谢JSPS向Shailendra Sharma和Shiveta Sharma提供的进行这项研究的财政援助。卡曼和J.D.G.琼斯由盖茨比基金会(英国)支持。

参考文献

  1. Ham,J.H.,Melanson,R.A。和Rush,M.C。(2011)。 伯克霍尔德氏菌:水稻的下一个主要病原体? < em> Mol Plant Pathol 12(4):329-339。
  2. Jeong,Y.,Kim,J.,Kim,S.,Kang,Y.,Nagamatsu,T.and Hwang,I。(2003)。 Toxoflavin   由引起谷物腐烂的伯克霍尔德氏菌产生 用于在许多田间作物中诱导细菌性枯萎。植物病 87(8):890-895。
  3. Kim,   J.,Kang,Y.,Kim,J.G.,Choi,O.and Hwang,I。(2010)。 发生 在韩国的水稻和大田作物上的Burlkholderia glumae。植物Pathol J 26(3):271-272。
  4. Sharma,S.,Sharma,S.,Hirabuchi,A.,Yoshida,K.,Fujisaki,K.,Ito,A.,Uemura,A.,Terauchi,R.,Kamoun,S.,Sohn,KH,Jones ,JD和Saitoh,H。(2013)。 部署伯克霍尔德氏菌 III型分泌系统作为一种有效的工具将病原体效应子易位到单子叶植物细胞。植物J 74(4):701-712。 
  5. Sohn,KH,Lei,R.,Nemri,A.and Jones,JD(2007)。霜霉病效应蛋白ATR1和ATR13促进拟南芥中的疾病易感性。 植物细胞在线19(12):4077-4090。
  6. Tsushima,S.,Naito,H。和Koitabashi,M。(1996)。 水稻细菌性谷物腐烂的致病因子假单胞菌(Pseudomonas glumae)的种群动态,在水稻植物的叶鞘上与该领域的疾病发展相关。 62(2):108-113。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Saitoh, H. and Terauchi, R. (2013). Burkholderia glumae Competent Cells Preparation and Transformation. Bio-protocol 3(23): e985. DOI: 10.21769/BioProtoc.985.
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raissa fedora
atma jaya catholic university
Can I use 2mm cuvvette for high competency method? What settings should I use?
8/31/2015 9:58:44 AM Reply
Hiromasa Saitoh
Tokyo University of Agriculture

We haven't used 2mm cuvette for high competency method. But you can try it according to the setting written in conventional method.

8/31/2015 7:06:49 PM


raissa fedora
atma jaya catholic university

I couldn't replicate the method using my Burkholderia isolate. The electroporator says arc detected, meanwhile the conventional method works fine. I wonder what's the possibility of failure during pulsing..

8/31/2015 10:33:45 PM