pPG612.1-mCherry 乳酸菌红色荧光报告表达载体 BioVector® pPG612.1-mCherry Lactic Acid Bacteria Fluorescent Reporter Vector

BioVector® pPG612.1-mCherry 乳酸菌红色荧光报告表达载体 BioVector® pPG612.1-mCherry Lactic Acid Bacteria Fluorescent Reporter 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® pPG612.1-mCherry 乳酸菌红色荧光报告表达载体

• 载体名称：pPG612.1-mCherry

• 质粒类型：乳酸菌-大肠杆菌广宿主游离型/整合型荧光报告穿梭载体

• 抗性基因：大肠杆菌选择抗性为氨苄青霉素 (Ampicillin, 100 μg/mL) 或红霉素；乳酸菌选择抗性为红霉素 (Erythromycin, 5–10 μg/mL) 或氯霉素（根据具体骨架衍生物而定，通常以红霉素 ermR 为主）

• 报告基团：mCherry 单体红色荧光蛋白 (Monomeric Red Fluorescent Protein)

• 复制子：大肠杆菌复制子 (pUC ori) + 乳酸菌广宿主低/高拷贝复制子（如 pSH71 或 pAMβ1 来源复制起点）

• 生物安全级别：1级 (BSL-1)，适合食品级或常规实验室改造使用

• 详细特征描述：BioVector® pPG612.1-mCherry 是一种专为食品级益生菌及乳酸菌属（包括 Lactococcus、Lactobacillus、Leuconostoc 等）设计的经典荧光报告与基因表达穿梭载体。该载体的核心是在广宿主原核表达骨架中，下游融合了经过密码子优化的 mCherry 单体红色荧光蛋白基因。mCherry 具有极为优异的光稳定性、快速成熟能力以及极低的细胞毒性。其最大激发波长为 587 nm，最大发射波长为 610 nm，可发出明亮的红色荧光。这不仅能完美避开乳酸菌培养基（如 MRS）及菌体自身产生的强大黄色/绿色自发荧光干扰，还非常适合与经典的 GFP 绿色荧光系统配合进行双色标记实验。研究者通常可将乳酸菌内源的结构性启动子（如 P32、P45 启动子）或诱导型启动子（如 Nisin 诱导的 PnisA 启动子系统）克隆至 mCherry 上游的多克隆位点（MCS），利用荧光信号实时监测基因转录活性，或进行目标蛋白的 C 端/N 端荧光融合表达示踪。

二、 细胞培养与质粒克隆条件

• 大肠杆菌克隆与扩增条件：进行重组子构建、外源启动子连接或质粒大量提取时，将质粒转化至常规大肠杆菌感受态细胞（如 DH5α 或 Top10）。使用标准 BioVector® LB 液体培养基或固体平板，添加相应抗生素（如 100 微克每毫升的氨苄青霉素或 150 微克每毫升的红霉素）。于 37°C 恒温振荡培养箱中以每分钟 200 转的转速孵育过夜。

• 乳酸菌转化与筛选条件：纯化后的重组质粒通过高压电转化法导入乳酸菌（如 L. lactis 或 L. plantarum）的高渗电转感受态细胞中。转化后的细胞需在 GM17 或 MRS 液体培养基中于 30°C 静态复苏 1.5 到 2 小时。随后涂布于含有 5 至 10 微克每毫升 BioVector® 红霉素的 GM17/MRS 固体琼脂平板上。乳酸菌的培养通常在 30°C 恒温培养箱中进行静态（厌氧或微需氧）孵育 24 至 48 小时。

三、 荧光监测与胞内影像学观察操作步骤

1. 种子液制备：挑取经测序验证正确的乳酸菌重组单菌落，接种于含有 5 微克每毫升红霉素的 MRS/GM17 液体培养基中，30°C 静态培养过夜。

2. 启动子激活与表达诱导：将过夜菌按 1:50 转接至新鲜培养基中。若使用 Nisin 诱导型系统（如 NICE 系统），在菌体生长至对数中期（OD600 约为 0.4–0.6）时，向体系中加入微量（如 1–5 ng/mL）的 BioVector® Nisin（乳链菌肽）开启转录；若是组成型启动子，则直接定期取样。

3. 荧光定量分析：吸取 200 微里不同生长阶段的菌液至黑壁无底 96 孔板中，利用多功能酶标仪进行检测（激发光 587 nm，发射光 610 nm）。计算“荧光强度/OD600”的比值，以精确评估乳酸菌在特定环境（如模拟胃肠道酸碱应激、胆盐压力）下的启动子转录动力学。

4. 显微成像观察：离心收集 1 毫升菌体沉淀，用无菌 PBS 缓冲液洗涤 2 次以彻底去除培养基成分引起的荧光背景。将菌悬液滴加于制备好的 1% 琼脂糖薄层载玻片上，在激光共聚焦显微镜或高级荧光显微镜下（使用 mCherry 或 TxRed 滤光片通道）观察红色荧光。这能清晰展示异源蛋白在乳酸菌胞内的局域化动态，或定性鉴定重组菌的标记效率。

四、 质粒与乳酸菌工程菌株的保藏技术

• 乳酸菌重组菌株冻存：将处于对数生长旺盛期的重组乳酸菌菌液，按照 7 比 3 的体积比与无菌的 BioVector® 细胞级甘油充分混合（最终甘油浓度为 30%）。混匀后立即分装于冻存管中，直接置于零下 80°C 超低温冰箱中冷冻保存。乳酸菌在 30% 甘油保护下可稳定存活数年以上。

• 质粒 DNA 长期储存：使用质粒提取试剂盒（由于乳酸菌细胞壁极厚，从乳酸菌提取质粒时需提前使用溶菌酶进行破壁处理；常规克隆建议直接从大肠杆菌扩增纯化）。将纯化后的质粒溶解于 BioVector® TE 缓冲液（pH 8.0）中，分装后置于零下 20°C 冰箱中冷冻保存，严禁反复冻融。

五、 质量控制与科研应用指南

• 质量控制标准：BioVector® pPG612.1-mCherry 载体经过严格的测序与功能性验证。通过全长高通量测序确认 mCherry 编码区、乳酸菌复制子及抗性选择标记序列 100% 正确；经检测不含外源核酸酶（DNase/RNase）污染。

• 核心实验应用方向：

  • 启动子探针表征：用于筛选和定量分析乳酸菌源的高强度结构性启动子或环境应激响应启动子。

  • 活体功能示踪：构建带有红色荧光标记的益生菌株，用于小鼠等动物胃肠道定殖动态、体内分布及定居寿命的活体成像（IVIS）与追踪。

  • 蛋白定位与多色成像：用于异源表达蛋白在乳酸菌细胞壁锚定、胞质定位或分泌途径的影像学示踪，可与表达 GFP 的载体联用。

  • 食品级发酵监控：在乳酸发酵、干酪成熟等工艺中，利用荧光信号无损、实时地监控益生菌的代谢与基因表达丰度。

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector® pPG612.1-mCherry Lactic Acid Bacteria Fluorescent Reporter Vector

• Vector Name: pPG612.1-mCherry

• Vector Type: Lactic Acid Bacteria (LAB) - E. coli Broad Host-Range Episomal Shuttle Vector

• Selection Marker: Ampicillin (100 μg/mL) or Erythromycin for E. coli; Erythromycin (5–10 μg/mL) for robust selection in Lactic Acid Bacteria (ermR determinant).

• Reporter System: Codon-optimized mCherry Monomeric Red Fluorescent Protein

• Replicon Infrastructure: E. coli pUC ori (high-copy in E. coli) + Broad host-range LAB replication origin (derived from pSH71 or pAMβ1).

• Biosafety Level: BSL-1 (Food-grade background compliant)

• Detailed Description: BioVector® pPG612.1-mCherry is a classic, high-performance fluorescent reporter shuttle vector tailor-made for genetic engineering, transcription profiling, and molecular tracking within Lactic Acid Bacteria (LAB), including Lactococcus lactis, Lactobacillus plantarum, and related probiotic strains. The functional core of this construct features a codon-optimized mCherry monomeric red fluorescent protein gene integrated into a broad-host shuttle backbone. Featuring a maximum excitation wavelength of 587 nm and a maximum emission wavelength of 610 nm, mCherry delivers exceptional photostability, swift maturation, and negligible cytotoxicity in prokaryotic cells. This red emission profile effectively bypasses the heavy yellow-green autofluorescence background characteristic of rich LAB media (such as MRS broth) and native bacterial aggregates, making it an ideal candidate for dual-color imaging alongside classic green fluorescent protein (GFP) platforms. Investigators can seamlessly insert native constitutive promoters (e.g., P32, P45) or inducible promoters (e.g., Nisin-controlled PnisA system) upstream of the multiple cloning site (MCS) to quantify transcriptional strength or forge C-terminal/N-terminal translational fusions to survey intracellular protein kinetics.

II. Culture Conditions and Cloning Parameters

• E. coli Propagation and Assembly Requirements: For traditional restriction cloning, Gibson assembly, or large-scale plasmid harvesting, transform the vector into standard E. coli competent cells (e.g., DH5α or Top10). Propagate clones using sterile BioVector® LB Liquid Medium or LB Agar plates supplemented with appropriate selection strain pressure (e.g., 100 μg/mL Ampicillin or 150 μg/mL Erythromycin). Incubate overnight in a shaking incubator at 37°C at 200 RPM.

• LAB Transformation and Environmental Benchmarks: Purified recombinant plasmids are introduced into LAB electrocompetent cells via high-voltage electroporation protocols. Post-pulse, cells must be recovered in hypertonic GM17 or MRS liquid broth at 30°C for 1.5 to 2 hours without agitation. Spread populations onto selection BioVector® MRS or GM17 Agar plates containing 5 to 10 micrograms per milliliter of BioVector® Erythromycin. Incubate plates at 30°C for 24 to 48 hours under static, microaerophilic, or anaerobic conditions.

III. Standardized Fluorescence Monitoring and Imaging Protocol

1. Inoculation and Seed Expansion: Pick a sequence-verified single colony of recombinant LAB harboring the pPG612.1-mCherry construct. Inoculate into sterile BioVector® MRS or GM17 medium containing 5 μg/mL Erythromycin and grow overnight at 30°C under static conditions.

2. Transcriptional Activation / Induction: Dilute the starter culture 1:50 into fresh selective medium. If using a Nisin-inducible framework (NICE system), supplement the broth with sub-lethal concentrations of BioVector® Nisin (typically 1–5 ng/mL) once the biomass density reaches mid-logarithmic phase (OD600 ~0.4–0.6) to switch on transcript cascade expression.

3. Fluorescence Quantification (Microplate Assays): Aliquot 200 microliters of the sampled cultures into a black-walled, clear-bottom 96-well microplate. Record absolute kinetic data using a multi-mode plate reader configured for excitation at 587 nm and emission at 610 nm. Standardize the promoter activity over time by rendering the "Fluorescence / OD600" ratio across simulated gastrointestinal stresses (e.g., low pH or bile salt challenges).

4. Microscopic Imaging and Visualization: Harvest 1 milliliter of the culture, spin at 5000 RPM for 2 minutes, and wash the cell pellet twice with sterile PBS to completely strip away autofluorescent media remnants. Mount 3–5 microliters of the washed bacterial suspension onto a microscope slide prepared with a 1% agarose pad. Image under a laser scanning confocal microscope or advanced epifluorescence microscope using an mCherry/TxRed filter set to trace single-cell spatial localization or validate tagging efficiencies.

IV. Plasmid Preservation and Long-Term Storage Methodology

• LAB Recombinant Stock Archiving: Collect validated recombinant LAB strains during their active exponential growth phase. Thoroughly mix 700 microliters of the fresh liquid culture with 300 microliters of sterile, BioVector® Cell-Grade Glycerol to establish a final concentration of 30% glycerol inside a sterile cryovial. Transfer directly into a minus 80°C ultra-low temperature freezer for multi-year preservation.

• Purified Plasmid DNA Storage: For routine downstream cloning, harvest high-purity plasmid DNA from E. coli intermediates using a standard kit. (Note: Extraction directly from LAB requires a prolonged lysozyme pre-treatment step due to their thick peptidoglycan cell walls). Dissolve the purified DNA pellet in sterile BioVector® TE Buffer (pH 8.0) and store in single-use aliquots at minus 20°C, strictly preventing repeated freeze-thaw degradation.

V. Quality Control and Research Application Guidelines

• Quality Control Standards: The BioVector® pPG612.1-mCherry vector undergoes rigorous quality control pipelines. Full-length Sanger sequencing verifies 100% accuracy across the mCherry open reading frame, multiple cloning site (MCS), LAB replication engine, and the erythromycin resistance determinant. The product is certified free from host genomic DNA background and active nucleases (DNase/RNase).

• Core Experimental Applications:

  • Promoter-Probe Profiling: Discovery, screening, and quantitative characterization of native high-yield constitutive or environment-responsive promoters in lactic acid bacteria.

  • In Vivo Probiotic Tracking: Developing bright red fluorescently labeled probiotic strains to trace their colonization dynamics, spatial distribution, and metabolic survival within animal gastrointestinal tracts using In Vivo Imaging Systems (IVIS).

  • Sub-cellular Localization: Visual tracing of heterologous antigens or therapeutic proteins engineered for cell-wall anchoring, cytoplasmic retention, or extracellular secretion pathways in LAB.

  • Fermentation Monitoring: Non-destructive, real-time tracking of biomass vitality and target gene expression abundance during dairy, cheese, or industrial lactic acid fermentation processes.

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