可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX
Summary
该研究描述了使用pMGX质粒系统在大肠杆菌中使用单个质粒的多个基因的T7介导的共表达的方法。
Abstract
多重蛋白质的共表达对于合成生物学，研究蛋白质 – 蛋白质复合物以及表征和利用生物合成途径来说日益重要。在该手稿中，描述了在可诱导T7RNA聚合酶的控制下使用高效系统来构建多基因合成操纵子。该系统允许许多基因从一个质粒同时表达。这里，一组四个相关载体pMGX-A，pMGX-hisA，pMGX-K和pMGX-hisK与氨苄青霉素或卡那霉素抗性选择性标记（A和K）和具有或缺少N-末端六组氨酸标签（他）被披露。使用该载体系统构建合成操纵子的详细方案与相应的数据一起提供，显示含有五个基因的基于pMGX的系统可以容易地构建并用于在大肠杆菌中产生所有五种编码的蛋白质。这个系统em和协议使研究人员能够在大肠杆菌中常规表达复杂的多组分模块和途径。
Introduction
多重蛋白质的共表达越来越重要，特别是在合成生物学应用中，其中必须表达多个功能模块1 ;在研究蛋白质 – 蛋白质复合物时，其中表达和功能通常需要共表达2,3 ;并且在表征和利用生物合成途径时，其中通路中的每个基因必须表达为4,5,6,7,8。已经开发出许多用于共表达的系统，特别是在宿主生物体大肠杆菌中 ，实验室重组蛋白表达的工作马9 。例如，具有不同选择标记的多个质粒可用于使用大量不同的ex压迫矢量10,11 。用于多重蛋白表达的单个质粒系统已经使用多个启动子来控制每个基因的表达10,12 ;合成操纵子，其中多个基因编码在单个转录物2,13上;或者在一些情况下是编码最终被蛋白水解处理的多肽的单个基因，产生所需的期望的蛋白质14 。
图1：显示多顺反子载体构建的pMGX工作流程。 pMGX系统为可诱导T7启动子控制下的合成操纵子的构建提供了一种灵活的，易于使用的策略。e.com/files/ftp_upload/55187/55187fig1large.jpg“target =”_ blank“>请点击此处查看此图的较大版本。
在该手稿中，描述了在诱导型T7RNA聚合酶（ 图1 ）的控制下使用高效系统来构建多基因合成操纵子。该系统允许许多基因从一个质粒同时表达。它基于质粒系统，原来称为pKH22，已被成功应用于许多不同的应用6,7,8 。在这里，扩增该质粒组包括四个相关载体：pMGX-A，缺少任何C-或N-末端标签的表达载体和氨苄青霉素抗性标记; pMGX-hisA，编码N-末端六组氨酸标签和氨苄青霉素抗性标记的表达载体; pMGX-K，表达载体l检测任何C-或N-末端标签和卡那霉素抗性标记物;和pMGX-hisK，编码N-末端六组氨酸标签的表达载体和卡那霉素抗性标记。在本研究中，使用pMGX系统（特别是pMGX-A）产生含有五个基因的多顺反子载体的方法与大肠杆菌中每种单独蛋白质的成功产生一起被证明。
Protocol
获得感兴趣的基因 设计合成基因。 优化大肠杆菌表达的基因序列。 从序列（AvrII，NdeI，EcoRI和XbaI）中除去任何有问题的限制性位点。 纳入克隆的限制性位点建议使用5'-NdeI位点和3'-EcoRI位点。必要时可以使用其他网站;指选择的质粒的多克隆区（ 图S1 -S4 ）。如果需要，包括用于Western印迹检测的5'或3'编?…
Representative Results
在这项研究中，目标是共同表达来自单个质粒的五种蛋白质。编码N-或C-末端六组氨酸标签的五密码子优化的合成基因片段商购。通过PCR扩增合成基因，并单独克隆到PCR平片载体中并进行测序。为了产生多顺反子质粒，首先将5个感兴趣的基因克隆到合适的pMGX质粒pMGX-A中。 图2显示除了用NdeI + EcoRI消化的pMGX-A之外的PCRBlunt- yfg1和PCRBlunt-yfg2（参见?…
Discussion
多个基因的共表达越来越重要，特别是在复杂的多基因代谢途径3,4,5的表征和重建中。 pMGX系统在大肠杆菌常规6,7,8中进行多基因共表达，并可以由不同的研究人员访问。在本研究中，显示了使用pMGX-A的单个质粒系统同时产生了5种目的蛋白质。目前可获得的许多共表达系统仅允许插入两个基因，产生双顺反子质粒例如pET Duet载体12 。如果需要添加?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到加拿大自然科学与工程研究理事会的支持。
Materials
| Enzymes | |||
| Alkaline Phosphatase, Calf Intestinal (CIP) | New England Biolabs | M0290S | |
| AvrII | New England Biolabs | R0174S | |
| EcoRI | New England Biolabs | R0101S | |
| NdeI | New England Biolabs | R0111S | |
| XbaI | New England Biolabs | R0145S | |
| Herculase II Fusion DNA Polymerase | Agilent Technologies | 600677 | |
| T4 DNA Ligase | New England Biolabs | M0202S | |
| Name | Company | Catalog Number | Comments |
| Reagents | |||
| 1 kb DNA ladder | New England Biolabs | N3232L | |
| 4-20% Mini-PROTEAN TGX Stain-Free Protein Gels | Bio-Rad | 456-8095 | |
| 50 x TAE | Fisher Thermo Scientific | BP1332-4 | |
| Agar | Fisher Thermo Scientific | BP1423-500 | |
| Agarose | Fisher Thermo Scientific | BP160-500 | |
| Ampicilin | Sigma-Alrich | A9518-5G | |
| BL21 (DE3) chemically comeptent cells | Comeptent cell prepared in house | ||
| B-PER Bacterial Protein Extraction Reagent | Fisher Thermo Scientific | PI78243 | |
| dNTP mix | Agilent Technologies | Supplied with polymerase | |
| Gel Extraction Kit | Omega | D2500-02 | E.Z.N.A Gel Extraction, supplied by VWR Cat 3: CA101318-972 |
| Glycine | Fisher Thermo Scientific | BP381-1 | |
| His Tag Antibody [HRP], mAb, Mouse | GenScript | A00612 | |
| Immobilon Western Chemiluminescent HRP Substrate | EMD Millipore | WBKLS0100 | |
| IPTG | Sigma-Alrich | 15502-10G | |
| LB | Fisher Thermo Scientific | BP1426-500 | |
| Methanol | Fisher Thermo Scientific | A411-20 | |
| Pasteurized instant skim milk powder | Local grocery store | No-name grocery store milk is adequate | |
| Nitrocellulose membrane | Amersham Protran (GE Healthcare Life Sciences) | 10600007 | Membrane PT 0.45 µm 200 mm X 4 m, supplied by VWR Cat #: CA10061-086 |
| Plasmid DNA Isolation Kit | Omega | D6943-02 | E.Z.N.A Plasmid DNA MiniKit I, supplied by VWR Cat #: CA101318-898 |
| pMGX | Boddy Lab | Request from the Boddy Lab Contact cboddy@uottawa.ca | |
| Primers | Intergrated DNA Technologies | Design primers as needed for desired gene | |
| Synthetic Gene | Life Technologies | Design and optimize as needed | |
| Thick Blot Filter Paper | Bio-Rad | 1703932 | |
| Tris base | BioShop | TRS001.1 | |
| Tween-20 | Sigma-Alrich | P9416-50ML | |
| XL1-Blue chemically competent cells | Comeptent cell prepared in house | ||
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| BioSpectrometer | Eppendorf | RK-83600-07 | |
| Gel box – PAGE | Bio-Rad | 1658005 | Mini-PROTEIN Tetra Vertical Electrophoresis Cell |
| Gel Imager | Alpha Innotech | AlphaImager EC | |
| Incubator-oven | Fisher Thermo Scientific | 11-690-650D | Isotemp |
| Incubator-shaker | Fisher Thermo Scientific | SHKE6000-7 | MaxQ 6000 |
| Personna Razors | Fisher Thermo Scientific | S04615 | |
| Power Pack | Bio-Rad | S65533Q | FB300 |
| Transilluminator | VWR International | M-10E,6W | |
| Thermocylcer | Eppendorf | Z316091 | Mastercycler Personal, supplied by Sigma |
| UV Face-Shield | 18-999-4542 | ||
| Waterbath | Fisher Thermo Scientific | 15-460-2SQ | |
| Western Transfer Apparatus | Bio-Rad | 1703935 | Mini-Trans Blot Cell |
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Cite This Article
Hassan, M. I., McSorley, F. R., Hotta, K., Boddy, C. N. Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX. J. Vis. Exp. (124), e55187, doi:10.3791/55187 (2017).