BioVector® pDG364 Bacillus subtilis Integration Vector / pDG364 枯草芽孢杆菌染色体整合型质粒载体

一 产品基本信息与分子生物学背景

• 载体名称：pDG364。

• 载体分类：枯草芽孢杆菌（Bacillus subtilis）整合型/穿梭型克隆载体。

• 质粒大小：约 6.0 kb。

• 骨架源起与设计背景：

  pDG364 是研究革兰氏阳性模式细菌——枯草芽孢杆菌（B. subtilis）基因表达与遗传调控极为经典的染色体整合型（Integration vector）穿梭质粒。该载体由著名的 Bacillus 遗传学专家开发，设计初衷是为了克服质粒在枯草芽孢杆菌中复制不稳定性（Segregational instability）的致命缺陷。pDG364 属于非复制型整合载体（Non-replicative integration vector）。它含有大肠杆菌复制子，但不含有能在枯草芽孢杆菌内自主复制的 Ori 序列。因此，当其通过转化进入枯草芽孢杆菌后，必须通过同源重组的方式“逼迫”自身携带的表达元件完整地嵌入到枯草芽孢杆菌的染色体基因组中，从而实现外源基因极其稳定的、单拷贝的永久表达。

• 核心顺式作用元件与图谱特征：

  • $amyE$ 同源重组侧翼序列（$amyE$ Flanking Homology Regions）：这是 pDG364 的核心构造。多克隆位点（MCS）和选择抗性标记被完整夹在 $amyE$-front（$amyE$ 基因的前端片段）与 $amyE$-back（$amyE$ 基因的后端片段）之间。通过这两段长约数几百碱基对的同源序列，质粒在进入枯草芽孢杆菌后，会与宿主染色体上的 $amyE$（编码 $\alpha$-淀粉酶）位点发生双交换同源重组（Double-crossover homologous recombination）。

  • 多克隆位点（MCS）：位于重组夹层内，提供独特的限制性内切酶切位点（如 BamHI, SalI, PstI, EcoRI 等），便于嵌入目标启动子、报告基因或目标蛋白编码序列。

  • 双选择抗性标记（Dual Selection Markers）：

    1. 氨苄青霉素抗性基因（$Amp^R$ / bla）：位于重组区段之外的骨架上，专门用于在大肠杆菌（E. coli）中扩增克隆时进行选择。

    2. 氯霉素抗性基因（$Cm^R$ / cat）：位于 $amyE$ 重组夹层内部。当下游的双交换整合成功发生后，该基因会随目标片段一同嵌入枯草芽孢杆菌染色体中，作为枯草芽孢杆菌阳性整合克隆的筛选指征。

  • 原核复制子：含有高拷贝的 pUC ori，仅在大肠杆菌内起作用，确保可在 E. coli 中进行常规、高效的质粒分子克隆和大量抽提。

二 核心科研价值与遗传学转化应用

pDG364 质粒在芽孢杆菌基因工程、合成生物学及工业酶制剂开发中具有核心立足点：

1. 外源基因的超高稳定性单拷贝整合表达：

   在枯草芽孢杆菌中，普通游离型质粒极易在没有抗生素压力的情况下发生丢失（Plasmid loss），不适合大规模工业发酵。利用 pDG364 将目标基因整合至染色体后，外源片段将伴随细菌基因组的复制而复制，在不加任何抗生素的复杂发酵液中亦能实现 100% 稳定遗传。

2. 淀粉酶失活表型打靶与顺式双交换验证（$amyE$ Blasting Assay）：

   重组片段整合进染色体后，会彻底破坏宿主原有的 $amyE$ 基因结构，导致枯草芽孢杆菌完全丧失分泌 $\alpha$-淀粉酶的能力。科研人员可通过经典的“淀粉平板碘液染色法”（淀粉酶转阴实验），极其直观地排除由于单交换（Single-crossover）导致的质粒整株嵌入，精准锁定真正的双交换染色体无痕整合株。

3. 枯草芽孢杆菌启动子强度调控与功能基因打靶：

   常用于在 $amyE$ 位点引入不同的诱导型启动子（如 $P_{spac}$、$P_{xyl}$）或报告基因（如 $lacZ$、$gfp$），用来在完全平行的染色体背景下，精确定量测定中枢基因网络在芽孢形成（Sporulation）和感受态（Competence）发育阶段的生化时空驱动特性。

三 实验室大肠杆菌扩增、枯草转导、表型筛选标准步骤

1. 质粒在大肠杆菌（E. coli）中的克隆与纯化

• 常规转化：将常规构建好的 pDG364 重组质粒通过热击法转化入大肠杆菌（如 DH5$\alpha$ 或 TOP10）感受态细胞中。

• 平板筛选：涂布于含有 100 $\mu$g/mL 氨苄青霉素（Ampicillin）的 LB 固体平板上，37 摄氏度培养过夜。

• 质粒抽提：挑取阳性单菌落进行液体扩增，使用标准碱裂解法质粒抽提试剂盒回收高浓度的环状重组质粒，测定纯度（$OD_{260}/OD_{280} = 1.8-1.9$），作为转化枯草芽孢杆菌的供体底盘。（注：整合至枯草染色体需要完整的双链环状质粒或经特定酶切切出含有重组夹层的线性片段）。

2. 枯草芽孢杆菌感受态转化操作（两阶段两步法，以经典 168 株为例）

由于 pDG364 无法在枯草体内自主复制，转化时对 DNA 的投入量及感受态效率要求较高。

1. 配制芽孢杆菌生长培养基：提前制备 SPI 培养基（富含高氨基酸与糖）与 SPII 培养基（限制性无机盐盐度培养基，用于诱导感受态启动）。

2. 两阶段培养：

   • 将枯草芽孢杆菌宿主（如 B. subtilis 168）接种于 SPI 培养基中，37 摄氏度剧烈振荡培养至对数生长末期（$\sim T_0$ 阶段，通常可见生长曲线趋于平缓）。

   • 按 1:10 稀释体积转接至预热的 SPII 培养基中，37 摄氏度、低速温和振荡培养 1.5 - 2 小时，此时细菌进入最佳感受态（Competence）窗口。

3. 质粒转导：吸取 0.5 - 1.0 $\mu$L 处于高浓度状态（$\ge 500\text{ ng}$）的 pDG364 重组质粒 DNA，加入到 100 - 200 $\mu$L 的枯草感受态细胞悬液中。

4. 孵育复苏：在 37 摄氏度摇床中以 100 rpm 温和摇育复苏 60 - 90 分钟，允许同源重组酶系统（RecA 通路）有足够的时间在染色体位点完成链置换与双交换剪切。

3. 枯草整合株的双重表型筛选与确证

1. 抗生素耐药初筛：

   将复苏后的枯草菌液以 10,000 rpm 离心 1 分钟，弃部分上清。重悬后均匀涂布于含有 5 $\mu$g/mL 氯霉素（Chloramphenicol）的 LB 固体平板上。置于 37 摄氏度培养 18 - 24 小时。（注：只有成功发生染色体同源重组嵌入、或者极少数发生质粒单交换非特异嵌入的菌株才能在此浓度氯霉素平板上存活）。

2. $\alpha$-淀粉酶失活（AmyE阴性）表型确证（核心质控点）：

   • 配制含有 1% 可溶性淀粉（Soluble Starch）的 LB 固体筛选平板。

   • 用无菌牙签挑取氯霉素平板上长出的枯草单菌落，点阵式接种到淀粉平板上，同时接种一株未转化的野生型枯草 168 作为阳性对照。37 摄氏度孵育过夜。

   • 碘液染色显色：向长有菌落的淀粉平板表面倾倒适量 卢戈氏碘液（Lugol's iodine solution），使其完全浸没培养基表面，静置 1 - 2 分钟后倒掉。

   • 结果判定：

     • 野生型对照（或未成功双交换整合的假阳性株）：由于能合成并向胞外分泌淀粉酶，会将菌落周围的淀粉水解。碘液染色后，菌落周围会自发显现出一圈高度清晰、透明的“褪色透明圈（Halos）”。

     • 正确的 pDG364 双交换染色体整合株：由于 $amyE$ 基因已被重组片段从中彻底截断破坏，无法产生淀粉酶。碘液染色后，菌落周围完全没有透明圈，整体呈现均匀致密的蓝黑色或紫褐色，即表现为 $AmyE^-$ 表型。

3. 分子确证：挑选表现为 $Cm^R$ 且 $AmyE^-$ 的核心克隆，提取枯草基因组（Genomic DNA），设计跨越 $amyE$ 侧翼边界的引物进行 PCR 测序验证，锁定完美的单拷贝染色体工程整株。

Part 2 English Section

I General Information and Cell Biological Background

• Vector Name: pDG364.

• Vector Classification: Chromosomal integration / shuttle cloning vector designed for Bacillus subtilis.

• Plasmid Size Scale: Approximately 6.0 kb.

• Backbone Origin and Engineering Background:

  The pDG364 vector is a widely used insertion chassis configured to evaluate gene expression landscapes and transcriptional circuitries within the Gram-positive model organism Bacillus subtilis. Developed by prominent Bacillus geneticists, it was engineered to circumvent segregational and structural plasmid instability inherent to autonomous replication vectors in Bacillus species.

  Crucially, pSP364 operates as a non-replicative integration vector. While it possesses a standard E. coli replication origin, it lacks a functional origin of replication (Ori) configured for Bacillus subtilis hosts. Consequently, upon transformation into a B. subtilis recipient, the plasmid cannot persist episomally; it is forced to undergo homologous recombination with the host genome to rescue its selection cargo, resulting in highly stable, single-copy, permanent genomic integration of target cassettes.

• Core Cis-Acting Elements and Map Characterization:

  • $amyE$ Homology Flanking Insertion Segments: This region represents the functional machinery of pDG364. The Multiple Cloning Site (MCS) and internal selection markers are locked between $amyE$-front and $amyE$-back sequences. These homologous flanking sequences guide a precise double-crossover homologous recombination event targeting the endogenous chromosomal $amyE$ locus (encoding native extracellular $\alpha$-amylase).

  • Multiple Cloning Site (MCS): Nestled inside the recombination cassette, it provides discrete, unique restriction endonuclease cutting boundaries (e.g., BamHI, SalI, PstI, EcoRI) to anchor external promoters, reporter segments, or open reading frames.

  • Dual Selective Antibiotic Elements:

    1. Ampicillin Resistance Gene ($Amp^R$ / bla): Located externally to the $amyE$ homology locus on the plasmid backbone; serves exclusively as a selectable marker during standard E. coli molecular cloning routines.

    2. Chloramphenicol Resistance Gene ($Cm^R$ / cat): Embedded within the internal $amyE$ recombination boundaries. Following a successful double-crossover integration sequence, this cassette embeds into the B. subtilis chromosome, serving as a reliable index for identifying positive B. subtilis clones.

  • Prokaryotic Replicon: Features a standard high-copy pUC ori, functioning exclusively inside E. coli hosts to permit efficient plasmid amplification and recovery.

II Strategic Research Value and Genetic Applications

The pDG364 plasmid is a fundamental tool for Bacillus-based genetic engineering, synthetic biology, and industrial enzyme production:

1. Ultra-Stable Singe-Copy Genomic Expression Matrix:

   Standard episomal vectors in B. subtilis face significant plasmid loss when cultured over extended generations without continuous antibiotic selection pressure, rendering them unsuitable for industrial fermentation scales. Integrating target expressions via pDG364 into the host chromosome ensures that the target sequence replicates alongside the host genome, providing 100% inheritance stability in complex fermenter environments without requiring antibiotics.

2. Definitive Verification of Double-Crossover Recombination via Amylase Disruption:

   Successful target insertion disrupts the endogenous chromosomal $amyE$ gene framework, abolishing the host's ability to synthesize and secrete active $\alpha$-amylase. Utilizing a simple starch-iodine assay ($AmyE$ phenotype test), investigators can exclude false-positive single-crossover integration events and confirm correct double-crossover single-copy genomic insertion.

3. Evaluating Promoters and Mapping Temporal Gene Networks:

   pDG364 is standardly utilized to introduce custom inducible promoter configurations (e.g., $P_{spac}$, $P_{xyl}$) or visual reporters ($lacZ$, $gfp$) directly into the $amyE$ target domain. This provides a clean genomic environment to measure the precise temporal and spatial expression kinetics of regulatory networks during the complex life cycles of spore development (sporulation) and competence development.

III Laboratory E. coli Propagation, Bacillus Transformation, and Phenotypic Screening Protocols

1. Vector Propagation and Purification inside Escherichia coli

• Transformation Sequence: Deliver the engineered recombinant pDG364 plasmid configuration into standard competent E. coli cells (such as DH5$\alpha$ or TOP10) via standard heat-shock parameters.

• Selection Parameters: Plate the transformation mixture onto solid LB agar matrices supplemented with 100 $\mu$g/mL Ampicillin and cultivate at 37 °C overnight.

• Plasmid Harvesting: Harvest verified single colonies into selective liquid broth and extract plasmid constructs via standard alkaline-lysis kits. Ensure purity checks align with clear parameters ($OD_{260}/OD_{280} = 1.8-1.9$) to provide high-quality template stocks for downstream Bacillus delivery. Note: Successful double-crossover chromosomal entry requires clean circular plasmids or linearized fractions encompassing intact flanking regions.

2. Bacillus subtilis Competence Transduction (Classic Two-Step Method)

Because pDG364 cannot propagate autonomously in B. subtilis, high DNA mass input combined with optimized competence preparation protocols is required.

1. Reagent Media Setup: Prepare sterile SPI growth media (nutrient-rich, amino acid-supplemented formulation) and SPII starvation media (low-salt, mineral-restricted matrix to force competence machinery activation).

2. Two-Phase Biomass Cultivation:

   • Inoculate the target B. subtilis recipient strain (e.g., standard B. subtilis 168) into pre-warmed SPI growth medium. Agitate vigorously at 37 °C until the biomass transitions into late log phase ($\sim T_0$ point, marked by a stabilization of growth kinetics).

   • Transfer this culture at a 1:10 dilution into pre-warmed SPII starvation media. Reduce agitation speeds slightly and cultivate at 37 °C for 1.5 - 2 hours to optimize the transformation window.

3. Plasmid Interfacing: Add 0.5 - 1.0 $\mu$L of concentrated, high-purity pDG364 recombinant vector DNA ($\ge 500\text{ ng}$) into 100 - 200 $\mu$L of the prepared competent Bacillus cell suspension.

4. Outgrowth / Recombination Phase: Incubate the transformation mixture at 37 °C with gentle shaking at 100 rpm for 60 - 90 minutes. This outgrowth interval allows the endogenous recombinase network (RecA pathways) sufficient time to perform strand exchange and execute the required double-crossover alignment.

3. Dual Phenotypic Identification and Chromosomal Screening Verification

1. Primary Antibiotic Selection Gate:

   Centrifuge the recovered Bacillus outgrowth mixture at 10,000 rpm for 1 minute, decant a portion of the supernatant, resuspend the pellet, and plate the solution uniformly onto solid LB agar plates supplemented with 5 $\mu$g/mL Chloramphenicol. Incubate at 37 °C for 18 - 24 hours. Note: Only cells that have integrated the chloramphenicol resistance gene into their chromosome via homologous recombination—or rare single-crossover events—will form colonies.

2. Confirmation of $\alpha$-Amylase Inactivation ($AmyE^-$ Phenotype Validation):

   • Prepare a fresh set of solid selective LB agar plates enriched with 1% soluble starch.

   • Using a sterile toothpick, patch chloramphenicol-resistant colonies onto the starch plate in a grid pattern. Ensure a wild-type untransformed strain (e.g., B. subtilis 168) is patched on the same plate to serve as a positive control for amylase activity. Incubate at 37 °C overnight.

   • Lugol's Iodine Developing Assay: Flooding the plate surface with an appropriate volume of Lugol's iodine solution until the solid medium is completely submerged. Allow it to react for 1 - 2 minutes, then discard the excess liquid.

   • Phenotypic Result Determination:

     • Wild-type Reference (or false-positive single-crossover clones): Retain an intact $amyE$ gene structure and continue to secrete functional amylase, which hydrolyzes surrounding starch molecules. Upon iodine development, these colonies will be surrounded by a sharp, clear, colorless halo.

     • Correct pDG364 Double-Crossover Recombinants: The endogenous $amyE$ open reading frame is disrupted by the integration cassette, abolishing amylase production. Upon iodine development, these colonies exhibit no clear zone, with the surrounding agar staining a uniform dark blue-black or deep purple color ($AmyE^-$ phenotype).

3. Molecular Architecture Validation: Harvest verified $Cm^R$ and $AmyE^-$ candidate clones, isolate genomic DNA (gDNA), and run PCR verification across the integration boundaries to confirm single-copy genomic insertion before establishing working cell banks.

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