BioVector® Pseudomonas aeruginosa PAO1-lux Bioluminescent Stable Strain / PAO1-lux 发光标记稳转菌株

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-lux 稳定生物发光标记菌株。

• 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，荧光 siderophore 铁载体）。

• lux 发光标记特性：

  • 利用染色体定点整合（如使用迷你转座子 Mini-Tn5 系统）或高稳定性低拷贝质粒载体，将来源于发光杆菌（Photorhabdus luminescens）的完整的自发光 luxCDABE 操纵子结构性导入 PAO1 基因组中。

  • 无需添加底物：该操纵子同时包含了负责产生发光底物（长链脂肪醛）的合成酶基因（luxCDE）和负责催化发光反应的荧光酶基因（luxAB）。因此，活菌在进行正常的细胞能量代谢时，即可自发产生波长约为 490 nm 的持续蓝绿色生物发光（Bioluminescence），其发光强度与体内的活菌数量、代谢活跃度呈严格的线性正相关。

• 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-lux 菌株将铜绿假单胞菌的强致病特征与实时无创体外/体内成像技术结合，极大简化了微生物学传统终点检测：

1. 体外生物膜发展动态监测（Biofilm Dynamic Imaging）：

   铜绿假单胞菌是研究生物膜（Biofilm）的标杆模型。PAO1-lux 在 96 孔板或流体微流控芯片内组装成坚固的生物膜时，利用高敏感性冷 CCD 倒置光学显微镜或多功能微孔板读数仪，可在完全不破坏生物膜完整性、无需添加任何显色染料的前提下，连续数十小时在线追踪生物膜自组装、成熟及对药物抵抗的实时发光动力学曲线。

2. 小动物体内活体感染示踪（In Vivo Small Animal Imaging）：

   在构建小鼠急性肺炎（Pneumonia）、烧伤创面感染（Burn wound infection）、角膜炎以及角膜慢性溃疡等动物模型时，通过活体生物成像系统（IVIS），科研人员可在不同时间点直接穿透小鼠皮肤或组织，无创、定量地读取局部菌群的发光相对强度（RLU），绘制体内活菌空间分布与定殖消长动力学图谱。这避免了在每个时间点处死小鼠并匀浆计数菌落（CFU）的传统破坏性流程。

3. 新型抗菌药物与反生物膜策略高通量筛选（HTS for Antimicrobials）：

   可直接用于各种全自动机器人平台，开展小分子抗生素、天然中药提取物、抗菌肽（AMPs）以及群体感应（Quorum Sensing, QS）抑制剂的高通量药敏筛选。发光值（RLU）的陡降可在一分钟内敏锐反映出药物对细菌细胞壁完整性或代谢能（ATP 生成）的毁灭性破坏，极其适合抗生素杀菌动力学曲线（Time-kill Curves）的高频采样。

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-lux 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（可选）：为了在长期连续传代中彻底杜绝 lux 操纵子因基因同源重组或转座子丢失而出现“发光沉默”或质粒流失，建议在常规扩增平板/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：卡那霉素（Kanamycin，工作浓度 50 ug/mL）、庆大霉素（Gentamicin，工作浓度 15 - 30 ug/mL）或四环素（Tetracycline，工作浓度 10 - 20 ug/mL），具体需根据具体构建批次的抗性标签添加。若仅进行一两代内的普通功能性实验或动物接种，可直接使用不加抗生素的 LB 培养基。

2. 冻干粉/冻存菌种复苏步骤

1. 将不含（或含对应选择抗性）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-lux 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 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）。铜绿假单胞菌生长极快，次日即可长出直径 1 - 2 mm、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. 日常传代与发光活性监测

• 发光检测：将长有菌落的平板或装有摇菌液的离心管直接置于全黑暗室中，肉眼由于生理极限很难直接看到（需极暗适应数分钟，且强度取决于构建启动子），建议使用微孔板发光仪、酶标仪的 Luminescence 通道，或利用实验室常见的凝胶成像系统（关闭激发光源，仅开启化学发光/Chemiluminescence 拍摄模式），即可捕捉到极为明亮、轮廓清晰的白色/浅蓝色光斑。

• 传代扩增：挑取平板上单个健壮发光的单菌落，接种至 5 - 10 mL 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 6 至 12 小时（对数生长旺盛期）。由于该菌代谢剧烈，若培养超过 18 小时进入平台期晚期或衰亡期，其发光强度（RLU/CFU 比值）会因细胞内 ATP 库的枯竭而大幅下滑。因此，用于体内实验接种或药物筛查的菌液，必须使用对数生长期的鲜活菌体。

4. 菌株长期冷冻保存

• 长期甘油冷冻法：采集处于对数生长中晚期（振荡培养约 8 - 10 小时，OD600 约在 0.6 - 1.0 之间）、自发光值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-lux 的活菌数量及 lux 操纵子的发光动力学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-lux Bioluminescent 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 fluorescent siderophore iron-chelator).

• lux Luminescent Transgenic Configurations:

  • Engineered via site-specific chromosomal integration (utilizing mini-transposon Mini-Tn5 platforms) or stable low-copy replicon vectors, inserting the complete autonomous luxCDABE operon cloned from Photorhabdus luminescens into the host chromosome.

  • No Exogenous Substrate Addition Dictated: This specialized metabolic operon sequentially couples both the substrate-generating fatty acid reductase complex genes (luxCDE, manufacturing long-chain aliphatic aldehydes) and the light-emitting heterodimeric luciferase catalytic engines (luxAB). Consequently, living cells undergoing normal electron transport and cellular respiration spontaneously emit continuous light centered at a wavelength of approximately 490 nm. The photon flux density aligns in a tight, rigorous linear correlation with viable cell density and metabolic ATP pool availability.

• 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-lux line elegantly bridges the hyper-virulent physiological armaments of the PAO1 archetype with non-invasive, real-time optical tracking technology, replacing destructive endpoint analysis:

1. Real-time In Vitro Biofilm Architectural Kinetic Imaging:

   Pseudomonas aeruginosa serves as the foundational benchmark organism for biofilm dynamics. When PAO1-lux structures dense, unyielding extracellular matrices within 96-well microplates or microfluidic flow cells, high-sensitivity cooled charge-coupled device (CCD) micro-imaging networks scan the layers. Investigators can plot long-term maturation, structural scaffolding shifts, and antibiotic penetrative clearing kinetics across hours without damaging the spatial biofilm anatomy or administering destructive chemical fluorophores.

2. Non-Invasive In Vivo Small Animal Homing & Infection Tracking:

   In experimental settings modeling murine acute pneumonia, severe burn wound infection matrices, or chronic keratitis pathways, the integration of an In Vivo Imaging System (IVIS) transforms data capture. Photons easily penetrate host skin and dense tissue structures, yielding clean, quantitative relative light unit (RLU) reads. Investigators can non-invasively track localized bacterial localization, tissue load velocity, and clearing timelines within the same animal cohort over days. This successfully obsoletes the classical mandatory sacrifice, tissue homogenization, and plate colony counting (CFU) loops at every single serial check-point.

3. High-Throughput Screenings (HTS) for Novel Antimicrobials & Anti-QS Targets:

   Fully compatible with robotic automation grids to survey extensive small-molecule chemical libraries, native botanical extracts, antimicrobial peptides (AMPs), or Quorum Sensing (QS) subversion inhibitors. A sharp decay in RLU output signals immediate disruption of cell wall integrity, collapse of the trans-membrane proton motive force, or sudden depletion of intracellular ATP pools. This makes the strain exceptional for capturing high-frequency data points required to map fine-structure Time-kill Curves.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Growth Medium and Selective Resistance

While PAO1-lux is highly adaptable across standard bacteriological media, maintaining directed selection pressure prevents phenotypic drift:

• 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 (Optional): To entirely mitigate against spontaneous excision or silencing of the integrated lux operon during extended serial propagation cascades, supplement standard cultivation stocks with appropriate selection pressures. Common engineered resistance labels incorporate Kanamycin (optimized at 50 ug/mL), Gentamicin (ranging from 15 - 30 ug/mL), or Tetracycline (at 10 - 20 ug/mL), depending strictly upon the specific vector architecture variant dispatched. Omit selection pressures entirely for the direct 1 to 2 cycles of propagation immediate to animal inoculations or fine-scale enzymatic drug assays.

2. Thawing and Revitalization Routine

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

2. Retrieve the PAO1-lux 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). Proliferation is rapid; dense, 1 - 2 mm diameter colonies manifesting flat profiles, serrated edges, diffuse blue-green pigmentation, and distinct metallic sheet luster will emerge by morning.

3. Subculturing and Luminescent Output Tracking

• Bioluminescence Verification: Place active agar cultures or liquid shaking tubes directly into a completely dark room or box. While human scotopic vision requires distinct minutes of absolute adaptation to catch the photon glow, standard molecular imaging architectures easily read it. Run a basic blot or gel documentation station, turning off all excitation UV/transilluminator sources and running a pure Chemiluminescence capture loop to map bright, crisp white/blue luminescent silhouettes mirroring the colony topology.

• Passaging Routine: Pick a highly luminescent, 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 6 to 12 hours (targeting peak log-phase density). Because Pseudomonas aeruginosa drives heavy metabolic cascades, pushing cultures past 18 hours into deep stationary or decline phases will cause RLU metrics to fall sharply due to ATP pool starvation. Always harvest active, mid-log phase bacterial cells to ensure optimal performance during downstream animal challenge models or fine drug screenings.

4. Cryopreservation Protocol

• Long-Term Cryo-Freezing Routine: Harvest active liquid cultures running precisely through their mid-to-late logarithmic growth window (approx. 8 - 10 hours of continuous orbital shaking, corresponding to an OD600 range of 0.6 - 1.0) when the specific RLU-per-cell profile peaks.

• 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 kinetic integrity of the lux operon remain structurally intact for years.

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