BioVector® pRN5101 温敏型自杀质粒载体 / BioVector® pRN5101 Temperature-Sensitive Suicide Plasmid Vector

通用定义BioVector® pRN5101 是一种专门设计用于革兰氏阳性细菌（特别是多粘类芽孢杆菌 Paenibacillus polymyxa，苏云金芽孢杆菌 Bacillus thuringiensis 等芽孢杆菌属物种）中进行精准基因敲除，基因置换以及定向诱变的复制温度敏感型（Temperature-Sensitive）自杀质粒载体。该载体基于经典的葡萄球菌来源的温敏型复制子 pE194ts 改造构建而成。

BioVector® pRN5101 在低稳温度（如 28 到 30 摄氏度）环境下能够随宿主细胞正常进行自主复制和遗传维持；然而，一旦将环境温度提升至高限非允许温度（如 39 到 42 摄氏度），其自带的温敏型复制起始蛋白将发生构象失活，导致质粒丧失复制能力而表现为自杀效应。由于无法自主复制的质粒在筛选压力下会被迅速清除，这逼迫质粒上克隆的同源臂与宿主细菌的染色体靶位点之间发生高频的同源重组，从而实现目标基因的高效敲除或整合。在复杂工业菌株代谢工程和合成生物学中，它是用于破译非模式革兰氏阳性菌未知基因功能的黄金分子工具盒。

BioVector® pRN5101 技术参数

1. 载体核心元件布局与复制机制

• 温敏型复制区 携带经典的 pE194ts 复制子。这是一种典型的滚环复制模式元件，其中负责启动复制的核心引物蛋白基因发生突变，使其三维空间结构对热极其敏感。

  • 允许温度（Permissive Temperature）28 到 30 摄氏度。在此温度下，温敏复制蛋白能够完美结合于质粒的复制起点，质粒以中等拷贝数稳定存在。

  • 非允许温度（Non-permissive Temperature）39 到 42 摄氏度。在此温度下，复制起始蛋白变性失效，质粒自主复制完全停滞，启动自杀逻辑。

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• 抗性选择标记 整合有高效的红霉素抗性基因（Erythromycin Resistance）以及卡那霉素抗性基因（Kanamycin Resistance）。这些抗性基因在类芽孢杆菌和芽孢杆菌中具有优异的普适性表达效率。

• 多克隆位点 提供多个独特的单酶切位点，专门用于高效拼接目标基因的上游同源臂与下游同源臂。

2. 敲除工作原理与温度调控路径

利用 BioVector® pRN5101 进行基因敲除通常遵循标准的双交换重组逻辑：

1. 载体构建 将目标靶基因的上下游同源臂片段分别克隆至 BioVector® pRN5101 的多克隆位点中。

2. 低温电转 在 28 摄氏度的允许温度下，通过电击转化法将质粒导入目标多粘类芽孢杆菌中，利用红霉素进行初级稳转株筛选。

3. 高温自杀与单交换整合 将阳性转化子移至 39 摄氏度的非允许温度下进行高压选择性培养，同时维持抗生素选择压力。此时，只有通过单重组将整个质粒强行整合进染色体靶位点的细胞才能存活。

4. 低温无药诱导双交换 将单交换株转回 28 摄氏度无抗生素的环境中传代培养。温敏复制子的重新激活会导致染色体结构不稳定，从而触发自发性的第二次同源重组（脱出剪切）。最终，约有一半的几率会发生双交换，将染色体上的原生靶基因彻底替换切除，实现无疤痕或抗性标记替换的精准敲除。

主要科研应用

1. 多粘类芽孢杆菌功能基因组学与抗菌物质挖掘

• 多粘菌素合成阻断 广泛应用于多粘类芽孢杆菌（如 SC2 菌株或 WLY78 菌株）的非核糖体肽合成酶基因簇（如 pmxE 基因）的定向阻断，通过观察突变株是否丧失抗菌活性，直接确证抗菌脂肽的生物合成通路。

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2. 固氮菌株固氮酶调控网络解析

• 固氮核心元件敲除 用于定向敲除类芽孢杆菌中的关键固氮功能基因（如 nifB 基因）或细胞周期全局氮源调控因子，以解析细菌在不同铵盐浓度环境下的固氮效率漂移机制。

技术指标简表

操作提示 在将携带构建片段的 BioVector® pRN5101 质粒转化入多粘类芽孢杆菌时，由于革兰氏阳性菌的细胞壁较厚，必须选用处于对数生长早期的细胞制备高浓度电转感受态。电击完成后，必须立刻加入预冷的 BioVector® Nutrient Broth 营养肉汤培养基，并在 28 摄氏度下温和振荡复苏复壮至少 2 到 3 小时，以确保温敏复制子和抗性标记在涂布抗性平板前建立稳固表达。

BioVector® pRN5101 Temperature-Sensitive Suicide Plasmid Vector

General DefinitionBioVector® pRN5101 is a replication temperature-sensitive suicide plasmid vector specifically engineered for precise gene knockout, gene replacement, and directed mutagenesis in Gram-positive bacteria, particularly Paenibacillus polymyxa and Bacillus thuringiensis. The architecture of this vector is derived from the classic Staphylococcus-sourced temperature-sensitive replicon pE194ts.

BioVector® pRN5101 replicates autonomously and undergoes stable genetic maintenance within host cells at a lower permissive temperature of 28 to 30 degrees Celsius. However, upon shifting the incubation temperature to a non-permissive thermal threshold of 39 to 42 degrees Celsius, the encoded temperature-sensitive replication initiator protein undergoes conformational denaturation, completely abolishing plasmid replication and activating the suicide effect. Forced by the selective elimination of non-replicating episomes, the plasmid drives high-frequency homologous recombination between its cloned homology arms and the host chromosome. In the metabolic engineering of complex industrial strains and synthetic biology, this vector stands as a gold-standard molecular tool for decoding the function of unknown genes in non-model Gram-positive diazotrophs.

BioVector® pRN5101 Technical Specifications

1. Vector Core Elements and Replication Kinetics

• Temperature-Sensitive Replicon Features the classic pE194ts replicon, which operates via a rolling-circle replication mechanism driven by a mutated initiator protein gene sensitive to thermal shifts.

  • Permissive Temperature 28 to 30 degrees Celsius. At this temperature, the replication protein binds efficiently to the origin of replication, maintaining the plasmid at a medium copy number.

  • Non-permissive Temperature 39 to 42 degrees Celsius. The replication initiator protein becomes inactive, entirely halting autonomous episomal replication and forcing host integration.

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• Selection Markers Integrated with robust Erythromycin Resistance and Kanamycin Resistance expression cassettes, which deliver exceptional selection efficiency across diverse Paenibacillus and Bacillus species.

• Multiple Cloning Site Provides unique single restriction enzyme digestion sites dedicated to the seamless insertion of upstream and downstream homology arms flanking the target locus.

2. Knockout Mechanism and Temperature Cycling Workflow

Gene disruption using BioVector® pRN5101 follows a standard double-crossover homologous recombination logic:

1. Vector Construction The upstream and downstream homology arms flanking the target gene are cloned into the multiple cloning site of BioVector® pRN5101.

2. Low-Temperature Electrotransformation The recombinant vector is electroporated into the target host under the permissive temperature of 28 degrees Celsius, followed by primary transformant selection using erythromycin.

3. High-Temperature Suicide and Single-Crossover Integration The verified positive transformants are shifted to the non-permissive temperature of 39 degrees Celsius under continued antibiotic pressure. At this juncture, autonomous replication ceases, and only cells that integrate the entire plasmid into the chromosome via a single crossover event survive.

4. Low-Temperature Antibiotic-Free Curing for Double Crossover The single-crossover strains are shifted back to 28 degrees Celsius in antibiotic-free media. Re-activation of the temperature-sensitive replicon creates structural instability on the chromosome, triggering a spontaneous second crossover event. This results in the excision of the plasmid backbone, leaving a clean, markerless gene deletion or seamless gene replacement.

Primary Research Applications

1. Functional Genomics and Antimicrobial Discovery in Paenibacillus polymyxa

• Polymyxin Biosynthesis Disruption Extensively utilized for targeted disruption of non-ribosomal peptide synthetase clusters such as the pmxE gene in Paenibacillus polymyxa strains like SC2 or WLY78. By monitoring the loss of antimicrobial activity in mutants, researchers directly validate lipid peptide biosynthetic pathways.

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2. Regulatory Networks in Diazotrophic Strains

• Core Nitrogen Fixation Knockouts Deployed to knock out core functional nitrogenase genes like nifB or global nitrogen regulators in Paenibacillus species to elucidate metabolic signaling and shifts in nitrogen fixation capacity under varying ammonium concentrations.

Technical Data Summary

Handling Note When transforming BioVector® pRN5101 harboring candidate inserts into Paenibacillus polymyxa, electrocompetent cells must be prepared from early log-phase cultures due to the rigid cell wall barrier characteristic of Gram-positive bacteria. Immediately following electroporation, rescue the cells with pre-chilled BioVector® Nutrient Broth and incubate at 28 degrees Celsius with gentle shaking for at least 2 to 3 hours to permit robust phenotypic expression of selection markers prior to plating on antibiotic agar.

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