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Preparation of Genomic Overexpression Library
基因组超表达库的制备   

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Abstract

This protocol is used to identify/select for Escherichia coli genes that, when overexpressed in Vibrio cholerae Hsp33 (hslO gene) deletion mutant, protect against oxidative heat stress and, by extension, against HOCl-mediated protein damage. In the referenced publication, I found that V. cholerae mutant lacking Hsp33 (in addition to its established role in HOCl stress) has severe temperature sensitive phenotypes on MacConkey plates (this phenotype is not found in E. coli ΔhslO mutant). I constructed genomic expression libraries from E. coli and transformed the libraries into V. cholerae ΔhslO mutant. I then searched for complementing E. coli proteins that rescue the temperature sensitive phenotype of V. cholerae ΔhslO mutant on MacConkey plate. This protocol might work for searching complementation of other gene deletion mutants that have a good selective phenotype.

Keywords: Genomic(基因组), Overexpression(overexpression), Library(图书馆)

Materials and Reagents

  1. Bacteria donor (E. coli MG1655 wild-type or the ΔhslO deletion)
  2. Bacteria recipient (Vibrio cholerae O395 ΔhslO deletion)
  3. GenElute Bacterial Genomic DNA kit (Sigma-Aldrich)
  4. Plasmid pBR322 (New England BioLab, linearized with BamH1)
  5. Plasmid pET11a (New England BioLab, linearized with BamH1)
  6. BamH1 (New England Biolabs)
  7. BfuCI (New England Biolabs)
  8. T4 DNA ligase (New England Biolabs)
  9. Gel Extraction Kit (QIAGEN)
  10. Ultracompetent XL10-Gold cells (Stratagene)
  11. Wizard Plus SV Minipreps kit (Promega Corporation)
  12. MacConkey plates (lab-made)
  13. LB medium
  14. TAE buffer

Equipment

  1. Gel electrophoresis apparatus
  2. Incubator for bacteria

Procedure

  1. Prepare genomic DNA from the donor E. coli strains (MG1655 wild-type or the ΔhslO deletion) from 5 ml overnight culture (in LB medium) using the GenElute Bacterial Genomic DNA kit following the manufacturer’s instruction.
  2. Partially digest the genomic DNA (2 μg) with 0.05-0.2 units of BfuCI in a total volume of 50 μl for 10 min at 37 °C. Then heat inactive the enzyme.
  3. Analyze digested products on 1% agarose gels (in TAE buffer) and make sure that fragment sizes ranging between 1 and 8 kb.
  4. Gel purify fragmented genomic DNA with Gel Extraction Kit (some researchers prefer to separate DNA fragments into bins, for example 1-3 kb, 3-5 kb and 5-8 kb).
  5. Ligate the DNA fragments (approximately 50-80 ng/μl) into linearized pBR322/BamH1 or pET11a/BamH1 plasmid vectors (approximately 80-100 ng/μl) using T4 DNA ligase overnight at 15 °C following the manufacturer’s instruction.
  6. To propagate overexpressing plasmids library, transform 1-5 μl of ligation results into ultracompetent XL10-Gold cells following the manufacturer's protocol.
  7. Count colony forming units (CFU) of transformants the next day and calculate the size of the libraries (1 x 105 CFU/ml of cell or more is good library size).
  8. Combine the transformants by scraping off all colonies from the library and purify the total plasmids using Wizard Plus SV Minipreps kit.
  9. Transform 1-3 μl of the E. coli genomic library plasmids into recipient Vibrio cholerae ΔhslO deletion.
  10. Plate the transformants on MacConkey agar and incubate for 24 h at 43 °C to select for clones that rescue the temperature sensitive phenotype of the V. cholerae ΔhslO mutant strain (this mutant cannot grow on MacConkey plate at 43 °C).
  11. To ensure the growth phenotype, re-streak transformants that formed healthy looking colonies on MacConkey plates and grew for 24 h at 43 °C.
  12. To eliminate the possibility of mutations in the strain background, purify and re-transform plasmids (that rescured phenotype) of transformants into V. cholerae ΔhslO mutant.
  13. To identify inserted E. coli gene(s), purify the plasmid and send for DNA sequencing.

Acknowledgments

The protocol described here is adapted from one reported previously (Wholey et al., 2012).

References

  1. Wholey, W. Y. and Jakob, U. (2012). Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae. Mol Microbiol 83(5): 981-991.

简介

该方案用于鉴定/选择大肠杆菌基因,当在霍乱弧菌Hsp33(hslO基因)缺失突变体中过表达时,其保护免受氧化热应激,并且通过延伸 ,对HOCl介导的蛋白质损伤。 在引用的出版物中,我发现了 霍乱弧菌突变体缺少Hsp33(除了其在HOC1应激中的确立的作用)在MacConkey平板上具有严重的温度敏感表型(这种表型在大肠杆菌中没有发现 >Δsh10突变体)。 我构建了来自E的基因组表达文库。 大肠杆菌并将其转化为 V。 霍乱△HslO突变体。 然后我搜索互补。 大肠杆菌蛋白质,其拯救了V的温度敏感表型。 霍乱弧菌ΔhslO突变体。 这个协议可能工作,寻找互补的其他基因缺失突变体有良好的选择性表型。

关键字:基因组, overexpression, 图书馆

材料和试剂

  1. 细菌供体(大肠杆菌 MG1655野生型或ΔhslO缺失)
  2. 细菌受体(霍乱弧菌 O395ΔhslO缺失)
  3. GenElute Bacterial Genomic DNA试剂盒(Sigma-Aldrich)
  4. 质粒pBR322(New England BioLab,用 Bam H1线性化)
  5. 质粒pET11a(New England BioLab,用 Bam H1线性化)
  6. Bam H1(New England Biolabs)
  7. BfuCI(New England Biolabs)
  8. T4 DNA连接酶(New England Biolabs)
  9. 凝胶提取试剂盒(QIAGEN)
  10. Ultracompetent XL10-Gold细胞(Stratagene)
  11. Wizard Plus SV Minipreps kit(Promega Corporation)
  12. MacConkey平板(实验室制造)
  13. LB培养基
  14. TAE缓冲区

设备

  1. 凝胶电泳仪
  2. 细菌孵化器

程序

  1. 准备来自供体的基因组DNA。 使用GenElute Bacterial Genomic DNA试剂盒,根据制造商的说明,从5ml过夜培养物(在LB培养基中)培养大肠杆菌菌株(MG1655野生型或ΔhslO缺失)。
  2. 用总体积为50μl的0.05-0.2单位的BfuCl在37℃下部分消化基因组DNA(2μg)10分钟。 然后加热使酶失活。
  3. 在1%琼脂糖凝胶(在TAE缓冲液中)分析消化产物,并确保片段大小在1至8 kb之间。
  4. 凝胶用凝胶提取试剂盒纯化片段化的基因组DNA(一些研究人员更喜欢将DNA片段分离成箱,例如1-3kb,3-5kb和5-8kb)。
  5. 将DNA片段(大约50-80ng /μl)连接到线性化的p BR322/em BamH1或em ET11a/ Bam H1质粒载体(约80-100ng /μl),使用T4DNA连接酶,在15℃温育过夜。
  6. 为了繁殖过表达的质粒文库,按照制造商的方案将1-5μl连接结果转化到超感受态XL10-Gold细胞中。
  7. 次日计算转化体的菌落形成单位(CFU),并计算文库的大小(1×10 5 CFU/ml或以上的细胞是良好的文库大小)。
  8. 通过刮除文库中的所有菌落并使用Wizard Plus SV Minipreps试剂盒纯化总质粒来组合转化体。
  9. 转换1-3微升的 E。大肠杆菌基因组文库质粒转化到受体霍乱弧菌ΔhslO缺失中。
  10. 将转化子平板在MacConkey琼脂上并在43℃下孵育24小时以选择拯救霍乱弧菌ΔhslO突变株的温度敏感表型的克隆(该突变体不能在43℃的MacConkey板上生长)。
  11. 为了确保生长表型,在MacConkey平板上形成健康看起来的菌落并在43℃生长24小时的重排转化体。
  12. 为了消除菌株背景中的突变的可能性,将转化体的质粒(其重新表型)纯化并重新转化为V。 霍乱ΔhslO突变体
  13. 识别插入的 E。 大肠杆菌基因,纯化质粒并进行DNA测序

致谢

此处描述的方案改变自之前报道的方案(Wholey等人,2012)。

参考文献

  1. Wholey,W.Y。和Jakob,U。(2012)。 Hsp33 通过保护延伸因子Tu抗氧化降解而赋予抗漂白性, em> Vibrio cholerae 。 Mol Microbiol 83(5):981-991。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wholey, W. W. (2012). Preparation of Genomic Overexpression Library. Bio-protocol 2(19): e271. DOI: 10.21769/BioProtoc.271.
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