BioVector® pDR111_Bga no NLS 枯草芽孢杆菌异源表达与基因组整合载体 BioVector® pDR111_Bga no NLS Bacillus subtilis Heterologous Expression and Integration Vector

第一部分：中文说明

一、 产品基本信息与详细特征描述

• 产品名称：BioVector® pDR111_Bga no NLS 枯草芽孢杆菌异源表达与基因组整合载体

• 载体名称：BioVector® pDR111_Bga no NLS

• 质粒类型：枯草芽孢杆菌表达与染色体整合穿梭载体 (B. subtilis Expression & Integration Vector)

• 抗性基因：大肠杆菌选择抗性为氨苄青霉素 (Ampicillin, 100 μg/mL)；枯草芽孢杆菌整合选择抗性为分枝高哌素/光谱霉素 (Spectinomycin, 100 μg/mL)

• 启动子区域：Pspank 或 Pspank(hy) 强诱导启动子（IPTG 可调控诱导系统）

• 整合位点：amyE 位点（通过双交换同源重组）

• 报告/融合标签：Bga（来自青霉菌或内源的 β-半乳糖苷酶衍生标签），且去除了核定位信号 (no NLS, No Nuclear Localization Signal)

• 生物安全级别：1级 (BSL-1)

• 详细特征描述：BioVector® pDR111_Bga no NLS 是一种专门针对枯草芽孢杆菌 (Bacillus subtilis) 开发的高级通用型整合表达载体。该质粒的设计核心是在大肠杆菌中可作为高拷贝质粒进行克隆与扩增（依靠其自带的 bla 标记和 pUC 复制子），但在导入枯草芽孢杆菌后，由于缺乏该宿主特异性的复制起点，其必须通过两侧的 amyE 基因同源翼（amyE-front 和 amyE-back）发生高效率的双交换（Double-crossover）同源重组。该过程将目的表达框及光谱霉素抗性基因精准嵌入宿主染色体的 amyE 位点，实现单拷贝基因组稳定集成，杜绝了工业发酵中因质粒丢失造成的表达波动。该载体集成了受 LacI 严密压制的 IPTG 诱导型强启动子，且融合了 Bga 报告基因表型标签，关键的是，该版本移除了核定位信号 (no NLS)。这使得表达出的融合蛋白或报告蛋白能够自由、均匀地分布在原核生物的细胞质（Cytoplasm）中，而不会发生异常的亚细胞空间聚集。这一改动极大优化了异源蛋白在细菌胞内的折叠动态，避免了由于人工定位序列引起的细胞毒性，是枯草芽孢杆菌胞内高效高质表达、发酵酶制剂改良及精细定量转录分析的理想分子利器。

二、 细胞培养与质粒克隆条件

• 大肠杆菌克隆与扩增条件：进行外源片段连接与重组构建时，可将重组子转化至标准大肠杆菌克隆感受态细胞（如 DH5α 或 Top10）。使用无菌配制的 BioVector® LB 液体培养基或 LB 固体琼脂平板，并在其中添加终浓度为 100 微克每毫升的 BioVector® 氨苄青霉素。置于 37°C 恒温振荡培养箱中以每分钟 200 到 220 转的转速孵育过夜。

• 枯草芽孢杆菌转化与筛选条件：纯化验证后的整合重组质粒，通过电转化或天然化学感受态方法导入枯草芽孢杆菌。转化后的复苏细胞需接种于添加了终浓度为 100 微克每毫升 BioVector® 光谱霉素的营养琼脂或 LB 固体平板上。培养箱环境参数严格控制在 37°C。

三、 枯草芽孢杆菌整合转录及表型鉴定步骤

1. 质粒提取与线性化：使用 BioVector® 高纯度无内毒素质粒提取试剂盒回收重组质粒。为了大幅度提高染色体双交换重组成功率，建议在转化前使用限制性内切酶切断载体的非同源主链区，进行质粒线性化处理。

2. 感受态转化与复苏：将线性化 DNA 加入处于感受态生长期末期的枯草芽孢杆菌中，微量混匀。在 37°C 下静态孵育 30 到 40 分钟使其摄取 DNA。随后加入适量预热的富集肉汤，置于摇床中 37°C 低速复苏 1 小时，以使光谱霉素抗性基因充分表达。

3. 抗性筛选：将复苏菌液均匀涂布在含有 100 微克每毫升光谱霉素的筛选平板上，置于 37°C 培养 18 到 24 小时，直至长出清晰的光谱霉素抗性单克隆。

4. amyE 位点灭活与 Bga 报告验证：由于双交换会导致宿主内源 amyE（α-淀粉酶）基因断裂，可挑取单克隆点样于含有 1% 可溶性淀粉的琼脂平板上，37°C 培养过夜，倾倒 BioVector® 碘液。若菌落周围未形成透明水解圈（淀粉不被水解），证明成功整合于 amyE 位点。同时，由于融合了 Bga（β-半乳糖苷酶活性衍生片段且 no NLS），可通过在含有 X-gal 且不含 NLS 干扰的平板上观察菌落呈均匀蓝色，来定性评估转录活性。

四、 质粒与工程菌株的保藏技术

• 大肠杆菌菌种冻存：将克隆有正确质粒的大肠杆菌接种扩增至对数生长中期（OD600 达到 0.6 左右）。吸取 700 微里菌液与 300 微里无菌的 BioVector® 细胞级甘油在无菌管中彻底混匀（最终甘油浓度为 30%），直接放入 零下 80°C 超低温冰箱长期保藏。

• 纯化质粒 DNA 储存：质粒 DNA 需溶解于无菌的 BioVector® TE 缓冲液（pH 8.0）或无核酸酶的超纯水中。分装成小体系后，置于 零下 20°C 冰箱中可稳定保存数年，避免频繁反复冻融。

五、 质量控制与科研应用指南

• 质量控制标准：BioVector® 出品的质粒经过严格的序列完整性及功能验证。通过高通量 Sanger 测序确认 Pspank 启动子区、LacI 结合位点、Bga 标记区及独特的 no NLS（无核定位信号） 关键突变区域全长序列 100% 准确无漂移；酶切物理电泳图谱与设计图谱完全相符；无宿主核酸及 DNase/RNase 酶污染。

• 核心实验应用方向：该整合载体主要用于枯草芽孢杆菌内异源重组蛋白的非聚集性、均匀胞质表达；用于需要排除核定位信号干扰的原核细胞亚细胞定位对照实验；在大肠杆菌-芽孢杆菌穿梭系统中对复杂启动子和转录调节因子进行体内定量滴定；以及构建染色体单拷贝、高度遗传稳定的工业级酶制剂生产菌株。

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector® pDR111_Bga no NLS Bacillus subtilis Heterologous Expression and Integration Vector

• Vector Name: BioVector® pDR111_Bga no NLS

• Plasmid Type: B. subtilis Expression & Chromosomal Integration Shuttle Vector

• Selection Marker: Ampicillin (100 μg/mL) for propagation in E. coli; Spectinomycin (100 μg/mL) for chromosomal integration selection in B. subtilis.

• Promoter System: Robust Pspank or Pspank(hy) IPTG-inducible promoter infrastructure.

• Integration Target: amyE locus (via double-crossover homologous recombination)

• Reporter / Fusion Tag: Bga (β-galactosidase derived marker) configured with no Nuclear Localization Signal (no NLS).

• Biosafety Level: BSL-1

• Detailed Description: BioVector® pDR111_Bga no NLS is an advanced, highly specialized expression and site-specific integration vector meticulously engineered for genetic manipulation within Bacillus subtilis. The structural design permits high-copy plasmid replication and routine cloning within standard E. coli hosts via its pUC origin and bla (Ampicillin) selection marker. Crucially, because it lacks a functional replication origin for Gram-positive hosts, the plasmid functions strictly as a suicide integration vector upon introduction into B. subtilis. Flanked by homologous segments matching the front and back regions of the native amyE gene, it drives a high-efficiency double-crossover event that seamlessly embeds the expression cassette and the companion Spectinomycin marker into the host genome. This single-copy chromosomal integration resolves segregational instability and expression leakage inherent to episomal plasmids. Controlled by a tightly repressed, IPTG-inducible promoter, the expression cassette features a Bga reporter gene deliberately engineered with no NLS. Eliminating the nuclear localization signal ensures that the synthesized fusion protein or reporter distributes evenly across the prokaryotic cytoplasm instead of forming artificial sub-cellular clusters or aggregates. This modification optimizes protein folding behavior, mitigates transport-associated toxicity, and serves as an exceptional tool for cellular tracking, promoter titration, and metabolic engineering.

II. Culture Conditions and Cloning Parameters

• E. coli Propagation and Cloning Requirements: For traditional insert ligation, sequence modification, or large-scale plasmid harvesting, the vector should be transformed into E. coli competent cells (e.g., DH5α or Top10). Propagate transformed cells using sterile BioVector® LB Liquid Medium or LB Agar plates supplemented with BioVector® Ampicillin at a final working concentration of 100 micrograms per milliliter. Incubate overnight in a temperature-controlled shaking incubator at 37°C with an agitation speed of 200 to 220 RPM.

• B. subtilis Selection and Environmental Benchmarks: Sequence-verified recombinant constructs are introduced into B. subtilis via chemical competence or electroporation protocols. Transformed populations must be selected on BioVector® Nutrient Agar or LB Agar plates containing a final concentration of 100 micrograms per milliliter of BioVector® Spectinomycin. Constantly maintain incubation parameters at 37°C.

III. Standardized B. subtilis Integration and Phenotypic Verification Protocol

1. Plasmid Harvesting and Linearization: Extract the recombinant plasmid DNA using a BioVector® High-Purity Endotoxin-Free Plasmid Extraction Kit. To significantly enhance the frequency of double-crossover homologous recombination over single-crossover events, linearize the plasmid DNA using a restriction endonuclease that cleaves uniquely within the non-homologous vector backbone prior to transformation.

2. Competent Cell Transformation and Outgrowth: Add the linearized DNA directly into a suspension of B. subtilis competent cells harvested at their optimal receptive stage. Mix gently and incubate statically at 37°C for 30 to 40 minutes to facilitate DNA uptake. Supplement the transformation mixture with pre-warmed recovery broth and agitate at low speed for 1 hour at 37°C to allow full phenotypic expression of the Spectinomycin resistance gene.

3. Selective Plate Plating: Spread the recovered outgrowth evenly onto selective plates supplemented with 100 micrograms per milliliter of Spectinomycin. Invert the plates and incubate at 37°C for 18 to 24 hours until robust single transformants materialize.

4. Amylase Disruption and Bga Reporter Validation: Successful double-crossover events disrupt the native amyE gene encoding alpha-amylase. Patch individual spectinomycin-resistant colonies onto a BioVector® Nutrient Agar plate containing 1% soluble starch and grow overnight at 37°C. Flood the surface with BioVector® Iodine Solution; colonies lacking a surrounding clear zone (starch remaining unhydrolyzed) initially confirm target integration at the amyE locus. Concurrently, the expression of the Bga tag (devoid of NLS constraints) can be evaluated qualitatively on plates supplemented with IPTG and X-gal, yielding a uniform blue colony phenotype confirming localized cytoplasmic reporter function.

IV. Plasmid Preservation and Long-Term Storage Methodology

• Host Bacterial Glycerol Stock Preparation: For long-term archiving of E. coli strains carrying the validated construct, cultivate the host culture into its active logarithmic growth phase (OD600 around 0.6). Thoroughly combine 700 microliters of the bacterial culture with 300 microliters of sterile, BioVector® Cell-Grade Glycerol to achieve a final concentration of 30% glycerol inside a sterile cryovial. Store directly inside a minus 80°C ultra-low temperature freezer.

• Purified Plasmid DNA Archiving: Store purified plasmid DNA dissolved in sterile BioVector® TE Buffer (pH 8.0) or nuclease-free ultra-pure water. Aliquot into small single-use batches and preserve at minus 20°C. Repetitive freeze-thaw cycles must be strictly avoided to prevent physical double-stranded DNA cleavage.

V. Quality Control and Research Application Guidelines

• Quality Control Standards: The BioVector® pDR111_Bga no NLS vector undergoes stringent quality validation. High-throughput Sanger sequencing guarantees 100% sequence accuracy across the Pspank promoter region, the LacI repressor binding domains, the Bga open reading frame, and the critical mutated no NLS boundary. Restriction enzyme digestion mapping confirms complete structural alignment with theoretical plasmid maps. The product is certified free from contaminating host chromosomal DNA and active nucleases (DNase/RNase).

• Core Experimental Applications: This specialized integration vector is widely applied for the non-aggregating, homogenous cytoplasmic expression of heterologous proteins in B. subtilis; as a negative control framework in sub-cellular localization studies to exclude artificial nuclear-targeting peptide signaling; for the in vivo quantitative titration and kinetic assessment of complex promoters and transcription factors; and for fabricating single-copy, genetically stable industrial production strains for high-yield enzyme manufacturing.

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