搜索

Promoter Orientation of Prokaryotic Phase-variable Genes by PCR
采用PCR进行原核细胞可变基因启动子定位   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

One major mechanism of phase variable gene expression in prokaryotes is through inversion of the promoter element for a gene or operon. This protocol describes how to detect the promoter orientation of a phase-variable gene by PCR. This protocol, including primer design, is specific to detection of the promoter orientations of hyxR, a LuxR-like response regulator in Extraintestinal Pathogenic Escherichia coli (ExPEC) isolates (Bateman and Seed, 2012); however, this protocol can be generalized to other organisms and genes to discriminate prokaryotic promoter inversions by PCR through size discrimination of the amplification products. Expression of hyxR is regulated through bidirectional phase inversion of the upstream promoter region mediated by a member of the family of site-specific tyrosine recombinases called Fim-like recombinases. The recombinases recognize inverted DNA repeat sequences flanking the promoter and produce a genomic rearrangement, orientating the promoter in favor or disfavor of gene expression.

Materials and Reagents

  1. Escherichia coli (E. coli) isolate UTI893
  2. Sterile distilled, deionized water (diH2O)
  3. Agarose, molecular biology grade, standard sieve
  4. Ethidium Bromide, 1 mg/ml in distilled, deionized water (diH2O) (or other agent to visualize DNA)
  5. Taq polymerase (1 U/μl) with 10x NH4 buffer (APEX Bioresearch Products, catalog number: 42-409 )
  6. 10 mM dNTP mix, PCR grade (Life Technologies, catalog number: 18427-013 )
  7. Tryptone
  8. Yeast extract
  9. NaCl
  10. Tris base
  11. Boric acid
  12. EDTA (pH 8.0)
  13. SDS
  14. Glycerol
  15. Xylene cyanol
  16. Bromophenol blue
  17. hyxR phase-specific primers, 100 μM stock solution [Integrated DNA Technologies (IDT)]
    5’ – ACTGATAATAACCAGAGGCTTCTT – 3’
    5’ – CAGTGATTAACTTTCGAACATATTG – 3’
    5’ – GCGAAAGTTAATCACTGGTATGACC – 3’
  18. Tris-Borate-EDTA (TBE) (10x stock) (see Recipes)
  19. 10x DNA Loading Dye (see Recipes)
  20. Luria-Bertani broth (LB) culture medium (Sigma-Aldrich, catalog number: L3022-250G (see Recipes)
  21. 2% TBE agarose gel with EtBr (see Recipes)
  22. 20 ml 10x DNA Loading dye6 (xylene cyanol/bromophenol blue) (see Recipes)

Equipment

  1. 37 °C Incubator for bacteria with aeration
  2. Thermal cycler (Bio-Rad Laboratories)
  3. Gel electrophoresis system (Owl Separation Systems)
  4. UV Transilluminator with photo documentation (Bio-Rad Laboratories)

Procedure

  1. Start 5 ml overnight culture of E. coli isolate UTI89 (Note 1) in LB at 37 °C with aeration. Culture can be started from a colony on an agar plate or from a -80 °C glycerol stock.
  2. Setting up the phase-PCR reaction
    1. Phase PCR was performed using a 1:1:1 mixture of 3 phase primers including 1) an anchor primer outside of the invertible region (primer 1); 2) a phase-specific primer for the OFF orientation (primer 2); and 3) a phase-specific primer for the ON orientation (primer 3).



      Reaction mixture (Note 2)
      1 μl overnight bacterial culture (template)
      2.5 μl of 10x APEX NH4 buffer (1x final concentration)
      0.5 μl of 1 unit/μl APEX Taq polymerase (0.5 unit final concentration)
      1.25 μl of 50 mM MgCl2 (2.5 mM final concentration)
      0.1 μl of 100 μM stock of each of 3 primers (0.4 μM final concentration of each primer)
      0.5 μl of 10 mM dNTP mix (0.2 mM final concentration)
      up to 25 μl total volume with sterile diH2O.
    2. Thermal cycler program:



  3. Make 50 ml volume of 2% agarose in 1x TBE (see below). Pour gel into casting apparatus.
  4. Cover gel completely with 1x TBE (running buffer).
  5. Mix 5-10 μl of PCR reaction with 0.5-1.0 μl 10x DNA loading dye (approx. 1x final concentration). Load into wells and run samples at 100-120 V for 45 min to 1 h.
  6. Visualize and photograph using a UV gel transilluminator.
  7. Mixed phase populations show two bands by PCR, corresponding to the OFF and ON promoter orientations. The OFF orientation gives a 196 bp amplicon vs. a 231 bp amplicon for the ON orientation.

Notes

  1. UTI89 is a prototypic ExPEC cystitis isolate that was obtained from an adult patient with cystitis and has been well described in the literature (Mulvey, 1998). Primers for this protocol were designed to UTI89 genomic sequence, and while ExPEC isolates are similar, their genomic sequence may not be exact. It is recommended that researchers check that the primers listed in this protocol will work with other ExPEC isolates.
  2. All PCR reactions were performed using APEX Taq polymerase and accompanying buffers distributed by Genesse Scientific, Inc (San Diego, California, USA). Thermal cycler parameters were based on the manufacturer’s recommendations. Taq polymerase or related thermostable polymerases would be expected to provide similar performance in this assay with minor modifications of the reactions.
  3. 1x TBE is what you will use to make your agarose gel as well as the buffer used to run the gel.
  4. Any commercially available DNA loading dye is appropriate. The recipe given is only a home-made suggestion. Other recipes available use different tracking dyes and/or density agents.

Recipes

  1. 1x LB
    Suspend 20 g in 1 L of distilled water
    Autoclave for 15 min at 121 °C
    Final concentration of components (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L)
  2. 1 L 10x TBE buffer stock (Tris-borate-EDTA)
    108 g Tris base
    55 g Boric acid
    40 ml 0.5 M EDTA (pH 8.0)
    diH2O to 1 L
    Stir until completely dissolved
  3. 1 L 1x TBE buffer (working) (Note 3)
    100 ml 10x TBE
    900 ml diH2O
    Mix well
  4. 2% TBE agarose gel with EtBr
    2 g agarose per 100 ml 1x TBE
    Heat in microwave (approx. 2 min) until agarose is dissolved
    Let stand at room temperature for 15-20 min to cool (you do not want to re-solidify)
    Add EtBr to a final concentration of 0.1 μg/ml just prior to pouring gel
  5. 20 ml 10x DNA Loading dye (Note 4) (xylene cyanol/bromophenol blue)
    Add 0.025 g each xylene cyanol and bromophenol blue
    1.25 ml 10% SDS
    12.5 ml 100% glycerol
    6.25 ml H2O
    Mix thoroughly and store at room temperature

Acknowledgments

This protocol is adapted from Bateman and Seed (2012).

References

  1. Bateman, S. L. and Seed, P. C. (2012). Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli. Mol Microbiol 83(5): 908-925.
  2. Mulvey, M. A., Lopez-Boado, Y. S., Wilson, C. L., Roth, R., Parks, W. C., Heuser, J. and Hultgren, S. J. (1998). Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science 282(5393): 1494-1497.

简介

在原核生物中相变基因表达的一个主要机制是通过逆转基因或操纵子的启动子元件。该协议描述了如何通过PCR检测相变基因的启动子方向。该方案包括引物设计,其特异性用于检测hyxR(在肠道致病性大肠杆菌(ExPEC)分离株中的LuxR样应答调节剂)的启动子定向(Bateman和Seed,2012);然而,该协议可以推广到其他生物体和基因以通过PCR通过扩增产物的大小鉴别来区分原核启动子逆转。 hyxR的表达通过由称为Fim样重组酶的位点特异性酪氨酸重组酶家族成员介导的上游启动子区的双向相位反转来调节。重组酶识别启动子侧翼的反向DNA重复序列并产生基因组重排,使启动子定向有利于或不利于基因表达。

材料和试剂

  1. 大肠杆菌(大肠杆菌)隔离UTI893
  2. 无菌蒸馏的去离子水(二H 2 O)
  3. 琼脂糖,分子生物学级,标准筛
  4. 溴化乙锭,在蒸馏的去离子水(diH 2 O)中1mg/ml(或其它试剂以显现DNA)
  5. 具有10x NH 4缓冲液(APEX Bioresearch Products,目录号:42-409)的Taq聚合酶(1U /μl)
  6. 10mM dNTP混合物,PCR级(Life Technologies,目录号:18427-013)
  7. 胰蛋白酶
  8. 酵母提取物
  9. NaCl
  10. Tris碱
  11. 硼酸
  12. EDTA(pH 8.0)
  13. SDS
  14. 甘油
  15. 二甲苯蓝
  16. 溴酚蓝
  17. hyxR相特异性引物,100μM储备液[Integrated DNA Technologies(IDT)]
    5'-ACTGATAATAACCAGAGGCTTCTT-3'
    5'-CAGTGATTAACTTTCGAACATATTG-3'
    5' - GCGAAAGTTAATCACTGGTATGACC-3'
  18. Tris-Borate-EDTA(TBE)(10x stock)(参见配方)
  19. 10x DNA加载染料(参见配方)
  20. Luria-Bertani肉汤(LB)培养基(Sigma-Aldrich,目录号:L3022-250G(参见Recipes))
  21. 2%TBE琼脂糖凝胶(含EtBr)(见配方)
  22. 20ml 10×DNA加载染料6(二甲苯蓝/溴酚蓝)(参见配方)

设备

  1. 37°C曝气用细菌孵育器
  2. 热循环仪(Bio-Rad Laboratories)
  3. 凝胶电泳系统(猫头鹰分离系统)
  4. 带有照片文件的UV透射仪(Bio-Rad Laboratories)

程序

  1. 开始5ml过夜培养E。大肠杆菌在37℃下通气的LB中分离UTI89(注释1)。培养可以从琼脂平板上的菌落或从-80℃甘油原液开始
  2. 设置phase-PCR反应
    1. 使用3相引物的1:1:1混合物进行相PCR,包括1)可逆区域外的锚定引物(引物1); 2)用于OFF取向的相特异性引物(引物2);和3)用于ON取向的相特异性引物(引物3)


      反应混合物(注2)
      1μl过夜细菌培养物(模板)
      2.5μl的10×APEX NH 4缓冲液(1x最终浓度)
      0.5μl1单位/μlAPEX Taq聚合酶(0.5单位最终浓度)
      1.25μl50mM MgCl 2(2.5mM终浓度)
      0.1μl每种引物的100μM储备液(各引物的终浓度为0.4μM)
      0.5μl10mM dNTP混合物(0.2mM终浓度) 用无菌diH 2 O使总体积达到25μl。
    2. 热循环程序:



  3. 制备50毫升体积的1%TBE中的2%琼脂糖(见下文)。 将凝胶倒入浇铸设备中。
  4. 用1x TBE(运行缓冲液)完全覆盖凝胶。
  5. 混合5-10μl的PCR反应与0.5-1.0μl10x DNA加载染料(约1x最终浓度)。 装入孔中,并在100-120V下运行样品45分钟至1小时
  6. 使用UV凝胶透照仪可视化和拍照
  7. 混合相群体通过PCR显示两条带,对应于OFF和ON启动子方向。相对于ON方向的231bp扩增子,OFF方向给出196bp扩增子

笔记

  1. UTI89是从膀胱炎的成年患者获得的原型ExPEC膀胱炎分离株,并且已经在文献中充分描述(Mulvey,1998)。该方案的引物设计为UTI89基因组序列,而ExPEC分离物是相似的,它们的基因组序列可能不是精确的。建议研究人员检查本协议中列出的引物是否能与其他ExPEC分离株一起使用
  2. 使用由Genesse Scientific,Inc(San Diego,California,USA)分配的APEX Taq聚合酶和伴随的缓冲液进行所有PCR反应。热循环仪参数基于制造商的建议。预期Taq聚合酶或相关的热稳定聚合酶在该测定中提供类似的性能,对反应进行少量修改
  3. 1x TBE是用来制作琼脂糖凝胶以及用于运行凝胶的缓冲液
  4. 任何市售的DNA加载染料是合适的。 给出的食谱只是一个自制的建议。 其他配方可使用不同的跟踪染料和/或密度剂

食谱

  1. 1x LB
    在1升蒸馏水中悬浮20克 在121℃下高压灭菌15分钟
    组分(胰蛋白胨10g/L,酵母提取物5g/L,NaCl 5g/L)的最终浓度
  2. 1L 10×TBE缓冲液储液(Tris-硼酸盐-EDTA) 108克Tris碱
    55克硼酸
    40ml 0.5M EDTA(pH 8.0)
    diH 2 O至1L
    搅拌至完全溶解
  3. 1 L 1x TBE缓冲区(工作)(注3)
    100 ml 10x TBE
    900ml diH 2 O v/v 混合良好
  4. 2%TBE琼脂糖凝胶,用EtBr
    2 g琼脂糖/100 ml 1x TBE
    在微波(约2分钟)中加热直到琼脂糖溶解
    让在室温下放置15-20分钟冷却(你不想重新凝固)
    在倾倒凝胶之前,加入EtBr至终浓度为0.1μg/ml
  5. 20ml 10×DNA加样染料(注4)(二甲苯蓝/溴酚蓝)
    加入0.025g二甲苯蓝和溴酚蓝
    1.25ml 10%SDS
    12.5ml 100%甘油 6.25ml H 2 O x / 充分混合并在室温下贮存

致谢

该协议改编自Bateman和Seed(2012)。

参考文献

  1. Bateman,S.L.和Seed,P.C。(2012)。 由肠外致病性大肠杆菌产生的亚硝化应激反应和细胞内巨噬细胞存活的表观遗传调节 Mol Microbiol 83(5):908-925。
  2. Mulvey,M.A.,Lopez-Boado,Y.S.,Wilson,C.L.,Roth,R.,Parks,W.C.,Heuser,J.and Hultgren,S.J。(1998)。 通过1型piliated uropathogenic大肠杆菌诱导和逃避宿主防御 。 Science 282(5393):1494-1497。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bateman, S. L. and Seed, P. (2012). Promoter Orientation of Prokaryotic Phase-variable Genes by PCR. Bio-protocol 2(20): e274. DOI: 10.21769/BioProtoc.274.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。