测绘细菌功能的网络和途径<em>大肠杆菌</em使用合成基因阵列
Summary
系统，大型合成基因（基因或基因上位性）互动屏幕可用于，探索遗传冗余和途径串扰。在这里，我们描述了一种高通量的定量合成基因阵列筛选技术，被称为ESGA我们制定的，阐明互作关系，并探索遗传相互作用网络<em>大肠杆菌</em>。
Abstract
表型是由一系列复杂的物理（ 例如，蛋白质-蛋白质）和功能（ 例如基因的基因或基因）的相互作用（GI）的1。虽然身体的相互作用可以表明，细菌蛋白相关复合，他们不一定透露途径级的功能relationships1的。 GI屏幕，其中带有两个缺失或失活的基因的双突变体的增长是衡量和相应的单突变体相比，可以照亮上位性位点之间的依赖关系，从而提供了一种查询和发现新的功能关系2。大型GI地图真核生物如酵母3-7，但GI信息仍稀疏，为原核生物8，阻碍了细菌的基因组功能注释。为此，我们和其他人开发的高通量定量细菌GI筛选方法9，10 </支持>。
在这里，我们提出所需的关键步骤进行定量E.大肠杆菌的合成遗传的阵列（ESGA）筛选程序，在基因组规模9，使用天然细菌接合和同源重组系统产生和测量的健身大量的双突变体在殖民地阵列格式。简单地说，一个机器人是用来传输通过结合，氯霉素（Cm） – 的突变等位基因，从设计HFR（高频重组）的“供体菌株的成有序的阵列卡那霉素（Kan）的 – 显着的F-受体菌。通常情况下，我们使用损失的，功能单一的非必需的基因缺失突变体轴承（ 例如 “庆应义塾”系列11）亚效等位基因突变和必需的基因（ 即基因赋予蛋白表达减少，稳定或活动9，12，13）协会非必要和必需的基因，水库查询功能pectively。共轭和随后通过同源重组介导的遗传交换后，将所得的双突变体包含两种抗生素的固体培养基上选择。后的产物，数字成像板和殖民地的大小进行定量计分使用一个内部的自动图像处理系统14。地理标志显示时的双突变体的增长速度是显着高于或低于预计9。加重（或负）的地理标志常常导致撞击上的相同的基本处理流程2的补偿途径，对基因的功能缺失型突变的之间。在这里，一个单一的基因被缓冲的损失，例如，可以是单突变体是可行的。然而，这两个途径是有害的损失和合成致死或疾病的结果（ 即增长缓慢）。相反，减轻（或正）的相互作用可以在同一个途径或蛋白复合物2作为基因之间发生删除单独任何一个基因的往往是足以扰乱途径或复合物，例如，额外扰动不降低活性，因此生长，进一步的正常功能。总体而言，系统地识别和分析GI网络可以提供公正，世界地图，大量的基因，错过了其他方法的途径级别的信息可以推断出9之间的功能关系。
Protocol
1。构建HFR Cavalli的供体突变株的重组工程15，16 用于构造的ESGA捐助污渍的步骤描述如下。简单地说，我们使用有针对性的λ – Red介导的同源重组所产生的PCR扩增可选择的DNA标记盒的片段创建非必需基因缺失突变株（1.1节），或者必需的基因亚效等位基因突变体供体株（1.2节），然后用16 “查询”定义GI网络。 注：</stro…
Representative Results
GIs reveal functional relationships between genes. Similarly, since genes in the same pathway display similar GI patterns and the GI profile similarity represents the congruency of phenotypes, we can group functionally related genes into pathways by clustering their GI profiles. Integrating GI and GI correlation networks with physical interaction information or other association data, such as genomic context (GC) relationships can also reveal the organization of higher-order functional modules that define core bio…
Discussion
我们提出了一个逐步的协议，，为使用机器人ESGA筛选，调查细菌的基因的功能，通过询问GI的途径。这种方法可以被用来研究单个基因，以及在大肠杆菌中的整个生物系统大肠杆菌 。小心地执行上述实验步骤，包括所有适当的控制措施，并进行认真分析和独立验证的GI的数据是新功能的发现成功的ESGA的关键方面。此外ESGA，在概念上类似的方法研究GI E.被称为GIANT大肠杆菌， <em…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是从基因组加拿大，安大略基因组研究所和加拿大卫生研究院拨款，JG和AE AG的资金支持，是一个收件人的凡尼尔加拿大研究生奖学金。
Materials
| I. Antibiotics | 2 | 36471 Remove |
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| Chloramphenicol | Bioshop | #CLR201 | 3 | 36472 Remove |
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| Agar | Bioshop | #AGR003 | 8 | 36481 Remove |
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| 3. Bacterial Strains and Plasmids | 9 | 36475 Remove |
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| Hfr Cavalli strain λred system (JL238) | Babu et al.14. | 10 | 36476 Remove |
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| pKD3 | E. coli Genetic Stock Centre, Yale | 11 | 36477 Remove |
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| Keio E. coli F- recipient collection | National BioResource Project (NBRP) of Japan11 | 12 | 36479 Remove |
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| Hypomorphic E. coli F- SPA-tag strains | Open biosystems; Babu et al.14 | 13 | 36491 Remove |
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| 4. Primers | 14 | 36486 Remove |
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| pKD3-based desalted constant primers | F1: 5′-GGCTGACATGGGAATTAGC-3′ R1: 5′-AGATTGCAGCATTACACGTCTT-3′ |
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| Desalted custom primers | Cm-R: 5′-TTATACGCAAGGCGACAAGG-3′ Cm-F: 5′- GATCTTCCGTCACAGGTAGG-3′ |
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| Desalted custom primers | F2 and R2: 20 nt constant regions based on pKD3 sequence and 45 nt custom homology regions F2 constant region: 5′-CATATGAATATCCTCCTTA-3′ R2 constant region: 5′-TGTGTAGGCTGGAGCTGCTTC-3’S1 and S2: 27 nt constant regions for priming the amplification of the SPA-Cm cassette and 45 nt custom homology regions S1 constant region: 5’AGCTGGAGGATCCATGGAAAAGAGAAG -3′ S2 constant region: 5′- GGCCCCATATGAATATCCTCCTTAGTT -3′ KOCO-F and KOCO-C: 20 nt primers 200 bp away from the non-essential gene deletion site or the essential gene SPA-tag insertion site |
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| Taq DNA polymerase | Fermentas | # EP0281 | 19 | 36487 Remove |
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| 10X PCR buffer | Fermentas | # EP0281 | 20 | 36488 Remove |
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| 10 mM dNTPs | Fermentas | # EP0281 | 21 | 36489 Remove |
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| 25 mM MgCl2 | Fermentas | # EP0281 | 22 | 36490 Remove |
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| Agarose | Bioshop | # AGA002 | 23 | 36492 Remove |
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| Loading dye | NEB | #B7021S | 24 | 36493 Remove |
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| Ethidium bromide | Bioshop | # ETB444 | 25 | 36497 Remove |
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| 10X TBE buffer | Bioshop | # ETB444.10 | 26 | 36494 Remove |
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| Tris Base | Bioshop | # TRS001 | 27 | 36495 Remove |
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| Boric acid | Sigma | # T1503-1KG | 28 | 36496 Remove |
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| 0.5 M EDTA (pH 8.0) | Sigma | # B6768-500G | 29 | 36498 Remove |
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| DNA ladder | NEB | #N3232L | 30 | 36499 Remove |
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| 6. DNA isolation and Clean-up Kits | 31 | 36500 Remove |
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| Genomic DNA isolation and purification kit | Promega | #A1120 | 32 | 36501 Remove |
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| Plasmid Midi kit | Qiagen | # 12143 | 33 | 36502 Remove |
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| QIAquick PCR purification kit | Qiagen | #28104 | 34 | 36512 Remove |
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| 7. Equipment for PCR, Transformation and Replica-pinning | 35 | 36503 Remove |
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| Thermal cycler | BioRad, iCycler | 36 | 36504 Remove |
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| Agarose gel electrophoresis | BioRad | 37 | 36505 Remove |
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| Electroporator | Bio-Rad GenePulser II | 38 | 36506 Remove |
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| 0.2 cm electroporation cuvette | Bio-Rad | 39 | 36507 Remove |
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| 42 °C water bath shaker | Innova 3100 | 40 | 36508 Remove |
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| Beckman Coulter TJ-25 centrifuge | Beckman Coulter | 41 | 36519 Remove |
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| 32 °C shaker | New Brunswick Scientific, USA | 42 | 36509 Remove |
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| 32 °C plate incubator | Fisher Scientific | 43 | 36510 Remove |
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| 250 ml conical flaks | VWR | # 29140-045 | 60 | 36529 Remove |
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| 15 ml sterile culture tubes | Thermo Scientific | # 366052 | 61 | 36530 Remove |
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| Rectangular Plates | Singer Instruments | 63 | 36532 Remove |
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| 96-well and 384-well microtitre plates | Singer Instruments | Nunc | 64 | 36533 Remove |
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| Plate roller for sealing multi-well | Sigma | #R1275 | 65 | 36535 Remove |
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| plates | ABgene | # AB-0580 | 66 | 36534 Remove |
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| Adhesive plate seals | Fisher Scientific | # 13-990-14 | 67 | 36537 Remove |
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| -80 °C freezer | Any Vendor | 68 | 36536 Remove |
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Tags
Bacterial Functional NetworksPathwaysEscherichia ColiSynthetic Genetic ArraysPhenotypesPhysical InteractionsFunctional InteractionsGene-gene InteractionsGenetic InteractionsGI ScreensEpistatic DependenciesFunctional RelationshipsLarge-scale GI MapsProkaryotesFunctional AnnotationBacterial GenomesQuantitative Bacterial GI Screening MethodsE. Coli Synthetic Genetic Array (eSGA) Screening ProcedureGenome-scale Screening
Cite This Article
Gagarinova, A., Babu, M., Greenblatt, J., Emili, A. Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays. J. Vis. Exp. (69), e4056, doi:10.3791/4056 (2012).