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Ultra-low Background DNA Cloning System
高效DNA克隆系统   

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Abstract

We have developed a method to clone DNA fragments into the E. coli plasmid vectors with almost 100% efficiency (Goto and Nagano, 2013). This method is based on highly efficient yeast-based in vivo cloning, and the subsequent cloning of the constructed plasmids into E. coli. Our method is useful for various applications: multifragment DNA cloning, cloning of large DNA fragments, and cloning into large plasmid vectors. Furthermore, the sites at which DNA fragments are joined are not always located at the restriction ends in the plasmid vector, thus making the cloning method more flexible. Our system does not require manipulation for assembling or joining DNA fragments in a test tube, the efficiency of which may sometimes depend on the reaction conditions or the skills of the person performing the procedure. Therefore, both success rate and efficiency are extremely high. However, our system has a disadvantage in that it requires 2 steps for transformation. Our method is an improved version of previously developed methods (Iizasa and Nagano, 2006; Nagano et al., 2007). Next figure shows the flowchart of our method.

Keywords: Plasmid(质粒), Vector(矢量), Gene manipulation(基因操作), Yeast(酵母), E. coli(大肠杆菌)


Figure 1. Flowchart of our method

Materials and Reagents

  1. Plasmids/nucleic acids
    1. Plasmid pSU32 (http://www.iac.saga-u.ac.jp/lifescience/su32/index.htm) (Figure 2)


      Figure 2. Plasmid pSU32

    2. E. coli plasmid vector containing the Ampr gene (bla gene) (e.g., pUC, pBluescript, and the other many types of commonly-used plasmid vectors)
    3. DNA fragment to be cloned
    4. PCR primers (the pSU30-14 and pSU30-23 pair for the preparation of the conversion cassette SU32, and primer pair for the preparation of the DNA fragment to be cloned)
      the primer pair for the preparation of the conversion cassette SU32
      pSU30-14
      5'-CAGGTGGCACTTTTCGGGGAAATGTG-3'
      pSU30-23
      5'-ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA

      AGTTTTAAATCAATCTAAAGTATATATGAGTAAACT-3'
      Example: Primer pair for the preparation of the DNA fragment to be cloned
      (in this case, the GFPuv gene was cloned into pUC19 plasmid. The crossover regions (the sequences to be joined) are underlined).
      pUCGFPF
      5'-GAGCGGATAACAATTTCACACAGGAAACAGCTATGGCTAGCA

      AAGGAGAAGAACT-3'
      pUCGFPR
      5'-TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTTATTTG

      TAGAGCTCATCCA-3'

  2. Kits
    1. (Optional) PCR Purification kit
      In our paper (Goto and Nagano, 2013), we used the MonoFas DNA Purification Kit I (GL Sciences, Tokyo, Japan). However, this kit currently does not work well in our laboratory. Instead, we are now using the NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL, Düren, Germany).
    2. Plasmid DNA purification kit for E. coli
      We used a Mini-M Plasmid DNA Extraction System (Viogene, Taipei, Taiwan) (Goto and Nagano, 2013).
    3. Suspension buffer (in the case of Mini-M Kit, we use 200 μl of MX1 buffer)

  3. Enzymes
    1. Proofreading DNA polymerase such as Prime STAR GXL DNA Polymerase (TAKARA BIO, Ohtsu, Japan)
    2. Restriction enzymes (New England Biolabs)

  4. Cells
    1. Yeast cells (commonly used strains containing the genotype trp1, such as YPH499)
    2. Electro-competent E. coli cells
      We used the E. coli HST08 Premium Electro-Cells (TAKARA BIO, Ohtsu, Japan) (Goto and Nagano, 2013). However, these cells currently do not work well in our laboratory. Instead, we are now making electrocompetent cells ourselves according to Molecular Cloning (4th edition, pages 177-182).

  5. Plates
    1. LB agar containing 100 μg/ml ampicillin (Molecular Cloning, 4th edition, page 1100)
    2. Synthetic complete plates lacking tryptophan (The Gietz Lab, University of Manitoba, http://home.cc.umanitoba.ca/~gietz/ or Gietz and Woods, 2002)
    3. Ampicillin (sodium salt)

  6. Others
    1. Plating beads
    2. Acid-washed glass beads (350-500 μm)
      We are making acid-washed glass beads ourselves. However, Acid-washed glass beads (425–600 μm) can be obtained from SIGAMA-ALDRICH (catalog number: G8772).
    3. Agarose gel (Molecular Cloning, 4th edition, pages 94-98)
    4. Materials and Reagents required for yeast transformation (The Gietz Lab, University of Manitoba, http://home.cc.umanitoba.ca/~gietz/ or Gietz and Woods, 2002)

Equipment

  1. Thermal Cycler
  2. Agorose gel electrophoresis device
  3. Centrifuge
  4. Optional: Picofuge
  5. Incubators (30 °C and 37 °C)
  6. Vortex machine
  7. Optional: FastPrep FP100A (MP Biomedical)
  8. Heat block (70 °C)
  9. Electroporation device and cuvette
  10. Polyacrylamide gel electrophoresis devices
  11. Glass beads (350-500 μm)

Procedure

  1. Preparation of conversion cassette SU32
    1. Prepare the conversion cassette SU32 by PCR using a plasmid pSU32 as template. To obtain this template, visit the following webpage.
      http://www.iac.saga-u.ac.jp/lifescience/su32/index.htm
    2. For PCR amplification, the primer pair was pSU30-14 and pSU30-23 (40 cycles of 98 °C for 10 sec, 55 °C for 15 sec, and 68 °C for 75 sec using Prime STAR GXL DNA Polymerase) (Goto and Nagano, 2013). When we conduct the PCR reaction, we use Prime STAR GXL DNA Polymerase, but you can use the other proofreading DNA polymerases.
      Note: Although we purified the amplified conversion cassette in our paper (Goto and Nagano, 2013), this purification step is not essential (purification is required for DNA quantification).
    3. Confirm the amplification by 1% agarose gel electrophoresis. The amplicon size is about 2.7 kb (2,651 bp) (Figure 3).


      Figure 3. Conversion cassette SU32

  2. Preparation of the linearized E. coli plasmid vector
    1. Digest the E. coli plasmid vector within the crossover region by using restriction enzyme.
      Notes:
      1. The required restriction ends can be located anywhere within the crossover regions of the vector. You can use both blunt-end restriction enzyme and sticky-end restriction enzyme, because restriction ends are not positions where joining reactions occur.
      2. This E. coli plasmid vector must contain the Ampr gene.
      3. PCR amplification of the E. coli plasmid vector is another way to prepare the linearized E. coli plasmid vector.
      4. Although we purified the digested plasmid in our paper (Goto and Nagano, 2013), this purification step is not essential.
    2. Confirm the digestion by 1% agarose gel electrophoresis.
    3. Next Figure shows one of the examples of the restriction digestion (Figure 4 also shows how to join the DNA fragments).


      Figure 4. pUC19 digested by EcoRI-HF and XbaI

  3. Preparation of the DNA fragment to be cloned.
    1. Amplify a DNA fragment of interest by PCR using proofreading DNA polymerase such as Prime STAR GXL DNA Polymerase.
      Notes:
      1. PCR primers should be designed to carry the sequences with more than 20 bp of homology to the crossover region. We often use the sequences with 30-40 bp of homology to the crossover region. For the highest efficiency, we recommend you to use PCR primers purified by polyacrylamide gel electrophoresis. However, the PAGE purification is not essential.
      2. Although we purified the DNA fragment of interest in our paper (Goto and Nagano, 2013), this purification step is not essential.
    2. Confirm the amplification by agarose gel electrophoresis.
  4. Transformation of DNA fragments into yeast.
    1. Transform these 3 DNA fragments into yeast by TRAFO protocol (Gietz and Woods, 2002). TRAFO protocol is available on the following webpage.
      http://home.cc.umanitoba.ca/~gietz/
      Notes:
      1. In our laboratory, we perform the transformation procedure at half or quarter scale of this method.
      2. The molar ratio of the 3 DNA fragments was 1:1:1. However, this ratio is not important (Goto and Nagano, 2013). (Ten-fold variation of the ratio probably does not affect the efficiency.) Required amount of the conversion cassette SU32 is < 50 ng.
    2. Select the transformants by incubating yeast cells on synthetic complete plates lacking tryptophan at 30 °C for 2 or 3 days.
  5. Purification of plasmid from yeast.
    Note: To purify plasmid DNAs from yeast, we use a Mini-M Plasmid DNA Extraction System, but you can use the other Plasmid DNA Extraction Kit for E. coli.
    1. Scrape all colonies from the plates with plating beads, and collect yeast cells by the centrifuge. Using the microfuge, centrifuge cells at 14,000 x g for 5 sec (We often use the picofuge at a maximum speed for 30 sec). Remove the supernatant.
    2. Add the suspension buffer (in the case of Mini-M Kit, we use 200 μl of MX1 buffer), and suspend the yeast cells by vortexing.
    3. Scoop equal amounts of acid-washed glass beads (350–500 μm) to the suspended solution using 1.5 ml tube, add glass beads into tube containing the suspended solution, and then disrupt yeast cells by shaking 3 times for 20 sec at speed 5.5 using the FastPrep FP100A. Alternatively, vortex 5 times for 1 min at maximum speed. Next pictures show these experimental steps.





    4. Add denaturation buffer (in the case of Mini-M Kit, we use 250 μl of MX2 buffer), and gently mix.
    5. Punch a hole in the bottom of the tube by needle (18 gauge). Next pictures show these experimental steps.









    6. Attach another tube to the bottom of the tube, and centrifuge them to recover the denatured solution in the bottom tube. Next pictures show these experimental steps.







    7. For subsequent procedures, follow the manufacturer’s instructions. However, use elution solution preheated to 70 °C. Because DNA concentration of the eluted solution is low, you cannot determine the concentration of DNA by agarose gel electrophoresis or spectrophotometer. However, the DNA concentration of the eluted solution is sufficient for next procedures. Therefore, the condensation of the eluted solution is not required.
  6. Pretreatment of the eluted solution (Optional)
    1. For the highest efficiency, digest 5 μl of the recovered plasmids (the eluted solution) with small amounts (e.g. 0.2 μl) of an appropriate restriction enzyme by directly adding the enzyme to the eluted DNA solution.
      Note: Because the principle of this method is complicated, read our paper (Goto and Nagano, 2013) carefully. Do not add an enzyme reaction buffer, because the salt may affect the next electroporation step.
    2. Incubate the tube at an appropriate temperature for more than 1 h.
  7. Transformation of the eluted solution into E. coli.
    Transform the eluted solution into E. coli electro-competent cells by electroporation and plate them on LB agar containing 100 μg/ml ampicillin.
  8. Analyze E. coli transformants by various methods.

Acknowledgments

This protocol was adapted from previously published paper, Goto and Nagano, (2013). Part of this work was supported by A-STEP (FS-stage) from the Japan Science and Technology Agency (JST) and by a Grant-in-Aid for Challenging Exploratory Research from the Japan Society for the Promotion of Science.

References

  1. Gietz, R. D. and Woods, R. A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350: 87-96.
  2. Goto, K. and Nagano, Y. (2013). Ultra-low background DNA cloning system. PLoS One 8(2): e56530.
  3. Iizasa, E. and Nagano, Y. (2006). Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/Escherichia coli shuttle vectors. Biotechniques 40(1): 79-83.
  4. Nagano, Y., Takao, S., Kudo, T., Iizasa, E. and Anai, T. (2007). Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation. Plant Cell Rep 26(12): 2111-2117. 

简介

我们已经开发了一种将DNA片段克隆到E中的方法。大肠杆菌质粒载体,几乎100%的效率(Goto和Nagano,2013)。该方法基于高效的基于酵母的体内克隆,以及随后将构建的质粒克隆到E中。大肠杆菌。我们的方法可用于各种应用:多片段DNA克隆,大DNA片段的克隆和克隆到大质粒载体中。此外,DNA片段连接的位点不总是位于质粒载体的限制性末端,因此使克隆方法更灵活。我们的系统不需要在试管中装配或连接DNA片段的操作,其效率有时可能取决于反应条件或执行该程序的人的技能。因此,成功率和效率都非常高。然而,我们的系统的缺点在于其需要2个步骤用于转化。我们的方法是以前开发的方法的改进版本(Iizasa和Nagano,2006; Nagano等人,2007)。下图显示了我们的方法的流程图。

关键字:质粒, 矢量, 基因操作, 酵母, 大肠杆菌


图1.我们的方法的流程图

材料和试剂

  1. 质粒/核酸
    1. 质粒pSU32( http://www.iac.saga-u。 ac.jp/lifescience/su32/index.htm )(图2)


      图2.质粒pSU32

    2. E。 包含Amp r 基因( bla基因)(例如,pUC,pBluescript和其他许多类型 常用质粒载体)
    3. 要克隆的DNA片段
    4. PCR引物(用于制备转化盒SU32的pSU30-14和pSU30-23对,以及用于制备待克隆的DNA片段的引物对)
      用于制备转化盒SU32的引物对
      pSU30-14
      5'-CAGGTGGCACTTTTCGGGGAAATGTG-3'
      pSU30-23
      5'-ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA

      AGTTTTAAATCAATCTAAAGTATATATGAGTAAACT-3'
      示例:用于制备要克隆的DNA片段的引物对
      (在这种情况下,将GFPuv 基因克隆到pUC19质粒中。将交叉区域(待连接的序列加下划线)。
      pUCGFPF
      5'-GAGCGGATAACAATTTCACACAGGAAACAGCTATG GCTAGCA

      AAGGAGAAGAACT-3'
      pUCGFPR
      5'-TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAG TTATTTG

      TAGAGCTCATCCA-3'

  2. 套件
    1. (可选)PCR纯化试剂盒
      在我们的论文(Goto和Nagano,2013)中,我们使用MonoFas DNA纯化试剂盒I(GL Sciences,Tokyo,Japan)。 然而,这个套件目前在我们的实验室中不能很好地工作。 相反,我们现在使用NucleoSpin凝胶和PCR清理工具包(MACHEREY-NAGEL,Düren,德国)。
    2. 用于大肠杆菌的质粒DNA纯化试剂盒
      我们使用了Mini-M质粒DNA提取系统(Viogene,台北,台湾)(Goto和Nagano,2013)。
    3. 悬浮缓冲液(在Mini-M Kit的情况下,我们使用200μlMX1缓冲液)

    1. 校对DNA聚合酶如Prime STAR GXL DNA聚合酶(TAKARA BIO,Ohtsu,Japan)
    2. 限制酶(New England Biolabs)

  3. 细胞
    1. 酵母细胞(通常使用的含有基因型 trp1 的菌株,如YPH499)
    2. 电容性大肠杆菌细胞
      我们使用大肠杆菌 HST08 Premium Electro-Cells(TAKARA BIO,Ohtsu,Japan)(Goto和Nagano,2013)。 然而,这些细胞目前在我们的实验室中不能很好地工作。 相反,我们现在根据分子克隆(4 th 版,第177-182页)制造电子竞争细胞。

    1. 含有100μg/ml氨苄青霉素的LB琼脂(Molecular Cloning,4版,第1100页)
    2. 缺乏色氨酸的合成完整平板(The Gietz Lab,University of Manitoba, http://home.cc.umanitoba .ca /〜gietz/或Gietz和Woods,2002)
    3. 氨苄青霉素(钠盐)

  4. 其他
    1. 电镀珠
    2. 酸洗玻璃珠(350-500μm)
      我们自己制造酸洗玻璃珠。 然而,酸洗玻璃珠(425-600μm)可以从SIGAMA-ALDRICH(目录号:G8772)获得。
    3. 琼脂糖凝胶(Molecular Cloning,4版,第94-98页)
    4. 酵母转化所需的材料和试剂(The Gietz Lab,University of Manitoba, http://home.cc .gitz和Woods,2002)

设备

  1. 热循环仪
  2. 琼脂糖凝胶电泳仪
  3. 离心机
  4. 可选:Picofuge
  5. 培养箱(30°C和37°C)
  6. 涡流机
  7. 可选:FastPrep FP100A(MP生物医学)
  8. 加热块(70℃)
  9. 电穿孔装置和试管
  10. 聚丙烯酰胺凝胶电泳仪
  11. 玻璃珠(350-500μm)

程序

  1. 转化盒SU32的制备
    1. 使用质粒pSU32作为模板,通过PCR制备转化盒SU32。要获取此模板,请访问以下网页。
      http://www.iac.saga-u.ac.jp /lifescience/su32/index.htm
    2. 对于PCR扩增,引物对是pSU30-14和pSU30-23(使用Prime STAR GXL DNA聚合酶,98℃10秒,55℃15秒和68℃75秒的40个循环)(Goto和Nagano,2013)。当我们进行PCR反应时,我们使用Prime STAR GXL DNA聚合酶,但您可以使用其他校正DNA聚合酶。
      br />
    3. 通过1%琼脂糖凝胶电泳确认扩增。扩增子大小约为2.7kb(2,651bp)(图3)

      图3.转换磁带SU32

  2. 线性化E的制备。大肠杆菌质粒载体
    1. 摘要。 通过使用限制性内切酶在交换区内的大肠杆菌质粒载体。
      注意:
      1. 所需的限制性末端可以位于载体的交叉区域内的任何地方。 您可以使用平末端限制酶和粘性末端限制酶,因为限制性末端不是发生连接反应的位置。
      2. 这种大肠杆菌质粒载体必须包含Amp sup基因。
      3. 大肠杆菌质粒载体的PCR扩增是制备线性化大肠杆菌质粒载体的另一种方法。
      4. 虽然我们在论文中纯化了消化的质粒(Goto和Nagano,2013),但这个纯化步骤不是必需的。
    2. 通过1%琼脂糖凝胶电泳确认消化
    3. 下图显示了限制性消化的一个实例(图4还显示了如何连接DNA片段)

      图4.通过EcoRI-HF和XbaI消化的pUC19

  3. 待克隆的DNA片段的制备。
    1. 使用校对DNA聚合酶如Prime STAR GXL DNA聚合酶通过PCR扩增感兴趣的DNA片段。
      注意:
      1. 应设计PCR引物以携带与交换区具有超过20bp同源性的序列。 我们经常使用与交叉区域具有30-40bp同源性的序列。 为了最高的效率,我们建议您使用通过聚丙烯酰胺凝胶电泳纯化的PCR引物。 然而,PAGE纯化不是必需的。
      2. 虽然我们在论文中纯化了感兴趣的DNA片段(Goto和Nagano,2013),但这个纯化步骤不是必需的。
    2. 通过琼脂糖凝胶电泳确认扩增。
  4. 将DNA片段转化到酵母中。
    1. 通过TRAFO协议将这3个DNA片段转化到酵母中(Gietz和Woods,2002)。 TRAFO协议可从以下网页获得。
      http://home.cc.umanitoba.ca/~gietz/
      注意:
      1. 在我们的实验室中,我们以该方法的一半或四分之一进行转换程序。
      2. 3个DNA片段的摩尔比为1:1:1。 然而,这个比例不重要(Goto和Nagano,2013)。 (比率的十倍变化可能不影响效率。)转换盒SU32的需要量< 50 ng。
    2. 通过将酵母细胞在缺乏色氨酸的合成完整平板上在30℃下温育2或3天来选择转化体。
  5. 从酵母中纯化质粒 注意:为了从酵母中纯化质粒DNA,我们使用Mini-M质粒DNA提取系统,但是可以使用其他质粒DNA提取试剂盒用于大肠杆菌。
    1. 用电镀小珠从平板上刮下所有菌落,并通过离心机收集酵母细胞。使用微量离心机,以14,000×g离心细胞5秒(我们经常使用picofuge以最大速度30秒)。除去上清液。
    2. 加入悬浮缓冲液(在Mini-M试剂盒的情况下,我们使用200μlMX1缓冲液),并通过涡旋悬浮酵母细胞。
    3. 使用1.5ml管将悬浮等量的酸洗涤的玻璃珠(350-500μm)加入到悬浮的溶液中,将玻璃珠加入含有悬浮溶液的管中,然后通过以速度5.5摇动3次20秒来破坏酵母细胞,使用FastPrep FP100A。或者,以最大速度涡旋5次,每次1分钟。下图显示了这些实验步骤。





    4. 加入变性缓冲液(在Mini-M Kit的情况下,我们使用250μlMX2缓冲液),轻轻混合。
    5. 通过针(18号)在管的底部打孔。 下图显示了这些实验步骤。









    6. 将另一个管附接到管的底部,并离心它们以回收在底部管中的变性溶液。 下图显示了这些实验步骤。







    7. 对于后续程序,请按照制造商的说明进行操作。然而,使用预热至70℃的洗脱溶液。因为洗脱溶液的DNA浓度低,所以不能通过琼脂糖凝胶电泳或分光光度计测定DNA的浓度。然而,洗脱溶液的DNA浓度足以用于下一步骤。因此,不需要洗脱溶液的冷凝。
  6. 洗脱溶液的预处理(可选)
    1. 为了最高效率,通过直接将酶加入到洗脱的DNA溶液中,用少量(例如0.2μl)合适的限制酶消化5μl回收的质粒(洗脱的溶液)。
      注意:因为这个方法的原理很复杂,请阅读我们的 纸(Goto和Nagano,2013)。 不要添加酶反应 缓冲液,因为盐可能影响下一个电穿孔步骤。
    2. 在适当的温度下孵育管1小时以上。
  7. 将洗脱的溶液转化为E。 大肠杆菌。
    将洗脱的溶液转化为E。 通过电穿孔将大肠杆菌电感受态细胞接种在含有100μg/ml氨苄青霉素的LB琼脂上。
  8. 分析。 大肠杆菌转化体

致谢

该协议改编自以前发表的论文,Goto和Nagano,(2013)。这项工作的一部分由来自日本科技机构(JST)的A-STEP(FS阶段)和来自日本科学促进会的挑战探索研究的助剂提供支持。

参考文献

  1. Gietz,R.D.and Woods,R.A。(2002)。 通过乙酸锂/单链载体DNA /聚乙二醇法转化酵母。 方法Enzymol 350:87-96
  2. Goto,K.and Nagano,Y。(2013)。 超低背景DNA克隆系统 PLoS One 8(2):e56530。
  3. Iizasa,E。和Nagano,Y。(2006)。 高效的基于酵母的体内 多个DNA片段的DNA克隆和同时构建酵母/大肠杆菌穿梭载体。 40(1):79-83。
  4. Nagano,Y.,Takao,S.,Kudo,T.,Iizasa,E。和Anai,T。(2007)。 通过一步转化将基于酵母的DNA片段重组到植物转化载体中。 Plant Cell Rep 26(12):2111-2117。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nagano, Y. and Goto, K. (2013). Ultra-low Background DNA Cloning System. Bio-protocol 3(16): e874. DOI: 10.21769/BioProtoc.874.
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