BioVector® Pseudomonas aeruginosa PAO1-GFP Fluorescent Stable Strain / PAO1-GFP 绿色荧光标记稳转菌株

一 产品基本信息与遗传学背景

• 菌株名称：铜绿假单胞菌（绿脓杆菌）PAO1-GFP 稳定绿色荧光标记菌株。

• 物种分类：细菌界（Bacteria），变形菌门（Pseudomonadota），$\gamma$-变形菌纲（Gammaproteobacteria），假单胞菌目（Pseudomonadales），假单胞菌科（Pseudomonadaceae），假单胞菌属（Pseudomonas）。

• 母本菌株背景（PAO1）：

  • PAO1 是全球公认、应用最广泛的铜绿假单胞菌标准模式参考株（Type Strain）。最初由澳大利亚墨尔本大学从人类伤口感染灶中分离获得，分子背景极其清晰。

  • 它是一种革兰氏阴性（Gram-negative）、兼性厌氧、专性需氧、具备单端极生鞭毛的高运动性杆菌。在常规有氧平板上生长旺盛，常自发分泌青脓素（Pyocyanin，蓝绿色高氧化还原活性毒素）和黄脓素（Pyoverdine，黄绿色荧光 siderophore 铁载体）。

• GFP 荧光标记特性：

  • 利用染色体定点整合（如使用 Mini-Tn7 转座子系统结构性整合至染色体单一中性位点 attTn7）或高稳定性低拷贝质粒载体，将绿色荧光蛋白基因（gfp，如增强型 egfp 或稳定型 gfp-mut2）导入 PAO1 基因组中。

  • 激发与发射波长：该绿色荧光蛋白受强启动子（如 tac, lac 或结构性表达的 PclpB 启动子）驱动。活菌在受到波长约为 488 nm 的蓝光或紫外光激发时，会放射出波长约为 507 - 510 nm 的强劲绿色荧光。荧光强度高，抗光漂白（Photobleaching）能力强。

• 生物安全级别：2级（BSL-2）。作为临床重要条件致病菌，活菌操作必须在二级生物安全柜内进行。

二 核心科研价值与转化医学应用

PAO1-GFP 菌株将铜绿假单胞菌的强致病特征与现代显微成像及流式分析技术结合，是空间组织学和单细胞行为学研究的利器：

1. 共聚焦显微镜下生物膜三维空间结构重塑（3D Biofilm Architecture）：

   铜绿假单胞菌是研究生物膜（Biofilm）的国际金标准。PAO1-GFP 在流体剪切力细胞（Flow Cells）或盖玻片表面组装成复杂生物膜时，利用激光共聚焦扫描显微镜（CLSM）进行 Z 轴层切扫描，可清晰重构出生物膜的三维“蘑菇状”空间微结构、水通道分布以及胞外基质包裹形态，无需后期荧光染色破坏。

2. 多菌种交互与协同定殖可视化（Polymicrobial Interactions）：

   在口腔菌斑、慢性伤口或肺部混合感染研究中，PAO1-GFP 常与带有红色荧光标记（如 mChrry/tdTomato）的其他条件致病菌（如金黄色葡萄球菌 S. aureus）共同孵育。利用多通道荧光显微镜，可以实时动态捕捉不同菌种在空间上的共定位、竞争性排斥、机械性缠绕以及群体感应（QS）介导的协同包裹行为。

3. 宿主细胞入侵、胞内寄生与吞噬动力学研究（Host-Pathogen Interactions）：

   常用于侵染体外培养的人巨噬细胞、肺泡上皮细胞（A549）或血管内皮细胞。通过流式细胞术（FACS）可高通量定量分析巨噬细胞对 PAO1-GFP 的吞噬吞吐率；利用荧光显微镜可清晰定位活菌在宿主细胞质内的定殖增殖、逃逸溶酶体降解的轨迹。

三 实验室菌株复苏、扩增传代与冷冻保存标准步骤

1. 扩增培养基与选择抗性配置

PAO1-GFP 表现出极为强健的营养适应性，但在日常培养中仍需注意维持其标记的遗传严谨性：

• 基础培养基：LB（Lysogeny Broth）肉汤/固体琼脂平板（推荐），或胰酪胨大豆（TSB/TSA）培养基。

• 选择性维持抗生素（关键，依具体构建而定）：为了在长期连续传代中彻底杜绝 gfp 基因因同源重组或转座子丢失而出现荧光流失，必须在常规扩增平板/肉汤中补充对应工程载体所带的抗生素。常见的选择压包括：庆大霉素（Gentamicin，染色体整合型常选用 15 - 30 ug/mL）、卡那霉素（Kanamycin，工作浓度 50 ug/mL）或四环素（Tetracycline，工作浓度 10 - 20 ug/mL）。如果是通过外源质粒维持，抗生素必不可少；若是染色体转座子定点整合型，短中期传代不加抗生素亦可稳定表达荧光。

2. 冻干粉/冻存菌种复苏步骤

1. 将含有对应选择抗性的 LB 固体平板提前置于常规培养箱或室温平衡，保持表面干爽。

2. 从超低温冰箱或液氮中取出 PAO1-GFP 的冻存管，置于冰上融化，或在生物安全柜内小心打开复苏安瓿管。

3. 用无菌移液管吸取约 200 - 500 uL 的常规无抗 LB 肉汤加入管内，轻柔吹打菌块使其完全悬浮溶解。

4. 吸取全量菌悬液，倾倒或用涂布棒密集涂布于选择性 LB 固体平板的浓集区域，随后使用接种环进行常规四区划线，以便挑取单菌落。

5. 将平板倒置置于 37 摄氏度普通有氧培养箱中，连续孵育 14 至 18 小时（过夜）。次日长出边缘微扁平、常带有特征性蓝绿色漫延和金属光泽的健壮菌落。

3. 日常传代与荧光活性监测

• 荧光检测：将长有单菌落的平板置于手提式紫外灯或凝胶成像系统的蓝光透射仪（Blue Light Transilluminator，约 470 nm）上，开启激发光，肉眼即可直接观察到大大小小的菌落爆发出极为耀眼、翠绿色的明亮绿色荧光（自发分泌的黄脓素在紫外下虽也有绿光，但 GFP 荧光具有极强的像素聚焦感且强度远超背景）。

• 传代扩增：挑取平板上单个绿色荧光饱满、边缘清晰的单菌落，接种至 5 - 10 mL 选择性液体 LB 肉汤试管中。置于 37 摄氏度、200 - 220 rpm 振荡摇床内，连续培养 10 至 14 小时（通常过夜摇菌）。为了开展精密成像或定量流式分析，建议选用对数生长中晚期的菌体，此时细菌活性最高，GFP 蛋白折叠最充分且荧光强度与菌落数成正比。

4. 菌株长期冷冻保存

• 长期甘油冷冻法：采集处于对数生长晚期、绿色荧光强度达到峰值的液体培养物。

• 冻存操作：在无菌分装管内，将 750 uL 液体菌悬液与 250 uL 灭菌高纯无菌甘油（或直接使用含 20% 甘油的专属保菌液）轻柔混匀，使最终甘油保护剂工作浓度达到 20% 左右。

• 保存条件：混匀后，立即将冻存管直接投入 -80 摄氏度 超低温冰箱，或者放入 液氮（-196 摄氏度） 蒸气层内。在此稳定的超低温物理状态下，PAO1-GFP 的活菌数量及 GFP 荧光发光活性可维持数年以上不发生衰减。

Part 2 English Section

I General Information and Genetic Architecture

• Organism Name: Pseudomonas aeruginosa PAO1-GFP Fluorescent Stable Transgenic Strain.

• Taxonomic Classification: Domain Bacteria, Phylum Pseudomonadota, Class Gammaproteobacteria, Order Pseudomonadales, Family Pseudomonadaceae, Genus Pseudomonas, Species Pseudomonas aeruginosa.

• Parental Strain Genomic Framework (PAO1 Reference Matrix):

  • PAO1 stands as the definitive, globally cross-referenced paradigm standard reference strain for Pseudomonas aeruginosa research. Originally recovered from a human wound site at the University of Melbourne, its complete genetic blueprint and physiological profiles are exhaustively mapped.

  • It manifests as a robust, Gram-negative, facultatively anaerobic, strictly aerobic-respiring motile bacillus equipped with a singular polar flagellum. It proliferates aggressively on standard media, spontaneously exuding Pyocyanin (a blue-green redox-active phenazine cytotoxin) and Pyoverdine (a yellow-green fluorescent siderophore iron-chelator).

• GFP Transgenic Integration Profiles:

  • Engineered via site-specific chromosomal integration (utilizing Mini-Tn7 transposon platforms structurally integrated into the unique single neutral attachment site attTn7 on the chromosome) or stable low-copy replicon vectors, introducing the green fluorescent protein gene (gfp, such as enhanced egfp or stable gfp-mut2) into the PAO1 genome.

  • Excitation and Emission Kinetics: Driven by strong constitutive or inducible promoters (e.g., tac, lac, or structural PclpB promoter matrices). Upon encountering an excitation blue light or UV laser peak centered near 488 nm, the active vegetative bacteria emit intense green fluorescence peaking at 507–510 nm. The transgenic profile yields high brightness and superb resistance against photobleaching.

• Biosafety Matrix: Designated under Biosafety Level 2 (BSL-2) containment guidelines. As a formidable opportunistic human pathogen associated with multi-drug resistant nosocomial flare-ups, all continuous handling tracks must be localized inside certified Class II Biosafety Cabinets.

II Strategic Research Value and Translational Fields

The PAO1-GFP line elegantly bridges the hyper-virulent physiological armaments of the PAO1 archetype with modern micro-imaging and cytometry workflows, serving as a powerful utility for spatial histology and single-cell tracking:

1. Confocal 3D Reconstitution of Biofilm Architecture:

   Pseudomonas aeruginosa serves as the foundational benchmark organism for biofilm dynamics. When PAO1-GFP structures complex extracellular frameworks in flow cells or onto glass coverslips, Laser Scanning Confocal Microscopy (CLSM) captures continuous optical Z-sections. Investigators can generate 3D volumetric renders modeling native "mushroom-shaped" macro-aggregates, internal fluidic water channels, and matrix organization without administering destructive exogenous counter-stains.

2. Visualizing Polymicrobial Spatial Interactions and Multi-Species Co-colonization:

   In setups modeling dental plaque development, chronic wound environments, or polymicrobial pulmonary pathology, PAO1-GFP is regularly co-cultured alongside distinct pathobionts tagged with red fluorescent options (e.g., mCherry- or tdTomato-labeled Staphylococcus aureus). Utilizing multi-channel fluorescence imaging, researchers can map fine-scale spatial co-localization, mechanical entanglement, competitive displacement, and quorum sensing (QS)-mediated symbiotic pooling over extended periods.

3. Tracking Cellular Intracellular Invasion, Endocytosis, and Macrophage Phagocytosis Kinetics:

   Extensively integrated to infection protocols involving cultured human macrophages, alveolar epithelial sheets (A549), or primary vascular endothelial cells. Flow cytometry (FACS setups) leverages the bright green marker to execute high-throughput quantification of macrophage phagocytic index metrics. Simultaneously, micro-imaging maps real-time intracellular colonization, vacuole division rates, and evasion trajectories away from host lysosomal degradation pathways.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Growth Medium and Selective Resistance

While PAO1-GFP is highly adaptable across standard bacteriological media, maintaining directed selection pressure prevents transgene drift or loss:

• Basal Matrix: Standard Lysogeny Broth (LB) liquid formulas or solid agar plates (Highly Recommended), or Tryptic Soy Broth/Agar (TSB/TSA) alternatives.

• Selective Maintenance Pressures (Critical, Variant Dependent): To entirely eliminate potential transgene excision or phenotypic fluorescence dimming across long-term serial propagation cascades, supplement cultivation stocks with specialized selection pressures. Common setups incorporate Gentamicin (frequently optimized at 15–30 ug/mL for chromosomal mini-Tn7 integrations), Kanamycin (at 50 ug/mL), or Tetracycline (at 10–20 ug/mL), depending strictly upon the specific engineering vector deployed. If utilizing a validated site-specific chromosomal integration line, short-to-mid term subculturing can proceed antibiotic-free without destabilizing green output.

2. Thawing and Revitalization Routine

1. Pre-warm selective LB agar plates inside a standard incubation suite or hold at room temperature until the surface moisture completely desorbs.

2. Retrieve the PAO1-GFP cryovial from ultra-low freezers or liquid nitrogen containment, and position on a chilled ice bed. If handling a lyophilized pellet, unseal the vacuum-packed glass ampoule inside a verified biosafety enclosure.

3. Dispense approximately 200–500 uL of sterile, antibiotic-free liquid LB broth directly into the vial, pipetting with smooth adjustments to completely resuspend and dissolve the cell mass.

4. Extract the uniform slurry and spread the dense volume onto the concentrated quadrant of the selective LB agar plate. Transition to a sterile loops or specialized single-use streaks to execute a classical four-quadrant streak pattern for single-colony segregation.

5. Invert the inoculated plates and house them within a standard 37 degree Celsius aerobic incubator for 14 to 18 hours (overnight). Dense, robust colonies manifesting flat profiles, diffuse blue-green pigmentation, and distinct metallic sheet luster will emerge by morning.

3. Subculturing and Fluorescent Output Tracking

• Fluorescence Verification: Position active agar plate cultures directly over a handheld UV lamp or a standard laboratory blue light transilluminator (~470 nm excitation spectrum). Upon activation, viable colonies will emit an immediate, intense, bright emerald-green fluorescent signature (while native Pyoverdine background glow also reacts under UV, intact GFP yields a distinctly focused, crisp, hyper-intense visual density completely overpowering background parameters).

• Passaging Routine: Pick a highly fluorescent, well-isolated single colony from the master plate and drop it into 5–10 mL of fresh selective liquid LB broth. Proliferate in a standard orbital shaking incubator calibrated to 37 degrees Celsius running at 200–220 rpm for 10 to 14 hours (typically an overnight run). To ensure optimal execution during high-resolution microscopic assays or quantitative cytometry (FACS), always harvest fresh cultures capturing cells in late-logarithmic phase density, where target protein folding strikes maximum saturation.

4. Cryopreservation Protocol

• Long-Term Cryo-Freezing Routine: Harvest active liquid cultures running precisely through their late-logarithmic growth window when the absolute green fluorescence per unit biomass tracks at peak saturation metrics.

• Glycerol Stock Stabilization: Inside a sterile cryovial, blend 750 uL of the active bacterial slurry with 250 uL of sterile, analytical-grade high-purity glycerol (or utilize pre-formulated 20% glycerol protective preservation media). Mix smoothly via gentle inversion to homogenize the suspension at a final 20% glycerol target cryoprotectant baseline.

• Storage Configuration: Seal the tubes tightly and store immediately inside an ultra-low -80 degree Celsius freezer, or plunge directly into liquid nitrogen (-196 degree Celsius) vapor storage. Under these hyper-chilled physical baselines, viable cell metrics and the green fluorescence structural integrity remain completely uncompromised for years.

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