伯氏疏螺旋体（Borrelia）伯氏疏螺旋体外转录测定的基本成分
Summary
体外转录测定可以破译伯氏疏螺旋体的转录调控机制。该协议描述了纯化伯氏疏螺旋体RNA聚合酶并进行体外转录反应的步骤。使用体外转录测定的实验方法需要对活性RNA聚合酶进行可靠的纯化和储存。
Abstract
伯氏疏螺旋体 是一种细菌病原体，代谢和基因组库有限。 伯氏双歧杆菌 在脊椎动物和蜱虫之间细胞外传播，并显着重塑其转录谱，以便在感染期间在不同的环境中存活。 伯氏疏螺旋体 研究的一个重点是清楚地了解细菌如何通过转录变化对其环境做出反应。 体外 转录测定允许对转录调节的基本机制进行生化解剖。在这里，我们提出了一个详细的方案，描述了 伯氏疏螺旋 体RNA聚合酶纯化和储存，西格玛因子纯化，DNA模板生成和 体外 转录测定。该协议描述了使用从 伯氏疏螺旋体 5A4 RpoC-HIS（5A4-RpoC）纯化的RNA聚合酶。5A4-RpoC是一种先前发表的菌株，在编码RNA聚合酶最大亚基的 rpoC 基因上携带10XHis标签。 体外 转录测定包括从菌株 5A4-RpoC 纯化的 RNA 聚合酶、管家西格玛因子 RpoD 的重组版本和 PCR 生成的双链 DNA 模板。虽然蛋白质纯化技术和组装体外转录测定的方法在概念上很好理解且相对常见，但RNA聚合酶的处理注意事项通常因生物 体 而异。此处介绍的方案设计用于伯 氏疏螺旋体 RNA聚合酶的酶学研究。该方法可用于测试转录因子、启动子和翻译后修饰对RNA聚合酶活性的作用。
Introduction
莱姆病和回归热是由疏螺旋体和疏螺旋体1，2，3 属的螺旋体病原体引起的。莱姆病是北美一种突出的媒介传播疾病，因此，伯氏疏螺旋体是研究螺旋体生物学的重要模式生物4，5。对B. burgdorferi转录调控机制的研究旨在更好地了解它在蜱载体和哺乳动物宿主之间循环时对环境变化的适应6，7。pH、温度、渗透压、营养可用性、短链脂肪酸、有机酸以及溶解氧和二氧化碳水平的变化调节了伯氏疏螺旋体在其节肢动物载体中存活和感染动物很重要的基因的表达8，9，10，11，12，13，14，15，16，17，18.将这些对刺激的反应与调节机制联系起来一直是伯氏疏螺旋体研究的一个重要方面19。
转录因子和西格玛因子控制执行细胞过程的基因的转录。莱姆病和回归热螺旋体含有一组相对稀疏的转录因子和替代西格玛因子。尽管如此，仍有复杂的转录变化指导伯氏双歧杆菌对环境的反应20，21，22。驱动伯氏双歧杆菌响应环境变化的转录变化的具体机制尚不清楚。体外转录测定是采用生化方法测定转录因子和西格玛因子的功能和调节机制的有力工具23，24，25，26。
最近建立了使用伯氏疏螺旋体RNA聚合酶的体外转录测定系统24。由于细菌通常具有独特的细胞生理学，不同物种和属的RNA聚合酶对酶纯化，酶储存和反应缓冲条件的反应不同27。B. burgdorferi在遗传上也与许多细菌物种相距甚远，其中RNA聚合酶已被研究20。酶制备的各个方面，如裂解、洗涤和洗脱缓冲液条件、储存缓冲液、体外转录反应缓冲液以及测定构建方法都会改变RNA聚合酶活性。在此，我们提供了一种用于纯化RNA聚合酶和sigma因子RpoD，生产线性双链DNA模板以及构建体外转录测定的方案，以促进使用该系统的实验室之间的可重复性。我们详细介绍了一个示例反应，以证明RpoD依赖性转录的线性范围，并讨论了该方法的局限性和替代方案。
Protocol
1. RNA聚合酶的纯化和RNA聚合酶原液的制备 使用先前描述的细胞收集方案 28，29 从 2-4 L 伯氏疏螺旋体 RpoC-His10X 中培养的 BSKII 培养基中培养的细胞沉淀 28，29。在500mL离心瓶中以10，000×g在4°C下离心30分钟，倒出上清液。 将细胞重悬于30 mL冰冷的HN缓冲液（10 mM HEPES，10 mM NaCl，pH 8.0）中。使?…
Representative Results
在 体外 转录反应中，反应的限制步骤是西格玛因子介导的转录起始，转录活性应随西格玛因子的量线性增加。我们介绍了 体外 转录实验的制备，测试一系列RpoD浓度以及两种浓度的RNA聚合酶，以观察放射性标记的核苷酸掺入RNA产物所产生的变化信号。显示了制备RNA聚合酶、RpoD和双链DNA模板储备液的代表性结果。代表性的 体外 转录反应可作为可接受的RNA聚合酶活性水平的一?…
Discussion
使用所提出的方案构建的体外转录测定最近用于研究转录因子在伯氏疏螺旋体中的作用，并可用于使用其他转录因子，sigma因子和分子23构建类似的实验。一旦获得来自伯氏疏螺旋体的活性RNA聚合酶并检测到其活性，就可以修改体外转录测定中的组分和条件。该测定具有高度的灵活性和可修改性。反应是模块化的，由冷冻原料制成，一旦选择了实验参数…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了克赖顿大学健康科学战略投资基金教师发展补助金的支持。B. burgdorferi RpoC-His10X菌株由蒙大拿大学的D. Scott Samuels博士慷慨提供。携带编码麦芽糖结合蛋白标记rpoD等位基因的pMAL-C5X质粒的大肠杆菌菌株由NIH落基山实验室的Frank Gherardini博士友好提供。
Materials
| 0.45 micron syringe filter | Thermo Scientific | 726-2545 | Step 1.7 and 2.3 |
| 50 mL conical tubes | MidSci | C50B | Step 1.3, and subsequent steps |
| 50 mL high-speed centrifuge tubes | Thermo Scientific | 3119-0050PK | Step 1.2 |
| 500 mL Centrifuge bottles | Thermo Scientific | 3120-9500PK | Step 1.1 |
| B-PER and instruction manual | Thermo Scientific | 78248 | Step 1.4 and 2.2 |
| Calcium chloride | Fisher Scientific | 10035-04-8 | Step 2.6 |
| Centrifugal filters 10 Kd cutoff | Millipore Sigma | UFC8010 | Step 1.11 and 2.11 |
| Cobalt resin and instruction manual | Thermo Scientific | 89969 | Step 1.9 |
| Dithiothreitol | Acros Organics | 426380500 | Step 1.4 and subsequent steps |
| Dnase (Nuclease) | Millipore Sigma | 70746 | Step 1.4 and 2.2 |
| Factor Xa Protease | Haematologic Technologies | HCXA-0060 | Step 2.6 |
| GE Typhoon 5 Phosphoimager | GE lifesciences | Multiple | Step 4.15 |
| Gel Imager | Bio-Rad | Mutiple | Step 1.13 and subsequent protien quality check steps |
| H2O for in vitro transcription | Fisher Scientific | 7732-18-5 | Step 3.2 and 3.3 |
| high fidelity PCR kit | New England Biolabs | M0530S | Step 3.1 |
| High-speed centrifuge | Eppendorf | Step 1.1, and subsequent steps | |
| HiTrap Heparin HP 5 x 1 mL | Cytiva Life Sciences | 17040601 | Step 2.8 |
| Imidazole | Sigma-Aldrich | 56750-100G | Step 1.9 |
| Lysozyme | Thermo Scientific | 90082 | Step 1.4 and 2.2 |
| Magnesium chloride | Fisher Scientific | S25401 | Step 4.1 |
| Manganese chloride | Fisher Scientific | S25418 | Step 4.1 |
| Mini protean tetra cell | Bio-Rad | Mutiple | Step 1.13 and subsequent protien quality check steps |
| NP-40 | Thermo Scientific | 85124 | Step 4.1 |
| NTP mixture | Thermo Scientific | R0481 | Step 4.1 |
| PCR purification kit | Qiagen | 28506 | Step 3.2 |
| PCR tubes | MidSci | PR-PCR28ACF | Step 1.12 |
| PD-10 Sephadex buffer exchange column and instruction manual | Cytiva | 17085101 | Step 1.10 and 2.10 (gel filtration column) |
| pMAL Protein Fusion and Purification System Instruction manual |
New England Biolabs | E8200S | Step 2.1 |
| Polyacrylamide gels AnyKD | Bio-Rad | 456-8125 | Step 1.13 and subsequent protien quality check steps |
| Potassium glutamate | Sigma-Aldrich | G1251 | Step 4.1 |
| Protease inhibitor | Thermo Scientific | 78425 | Step 1.4 and 2.2 |
| Radiolabeled ATP | Perkin Elmer | BLU503H | Step 4.2 |
| RNA Loading Dye, (2x) | New England Biolabs | B0363S | Step 4.13 |
| Rnase inhibitor | Thermo Scientific | EO0381 | Step 4.1 |
| Spectrophotometer | Biotek | Mutiple | Step 1.13 and subsequent protien quality check steps |
| TBE-Urea gels 10 percent | Bio-Rad | 4566033 | Step 4.14 |
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Diesen Artikel zitieren
Boyle, W. K., Sorensen, H. N., Bourret, T. J. Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays. J. Vis. Exp. (185), e64134, doi:10.3791/64134 (2022).