BioVector? Pseudomonas aeruginosa PAO1-GFP Fluorescent Stable Strain / PAO1-GFP 綠色熒光標(biāo)記穩(wěn)轉(zhuǎn)菌株

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 菌株名稱：銅綠假單胞菌（綠膿桿菌）PAO1-GFP 穩(wěn)定綠色熒光標(biāo)記菌株。

• 物種分類：細菌界（Bacteria），變形菌門（Pseudomonadota），$\gamma$-變形菌綱（Gammaproteobacteria），假單胞菌目（Pseudomonadales），假單胞菌科（Pseudomonadaceae），假單胞菌屬（Pseudomonas）。

• 母本菌株背景（PAO1）：

  • PAO1 是全球公認、應(yīng)用最廣泛的銅綠假單胞菌標(biāo)準(zhǔn)模式參考株（Type Strain）。最初由澳大利亞墨爾本大學(xué)從人類傷口感染灶中分離獲得，分子背景極其清晰。

  • 它是一種革蘭氏陰性（Gram-negative）、兼性厭氧、專性需氧、具備單端極生鞭毛的高運動性桿菌。在常規(guī)有氧平板上生長旺盛，常自發(fā)分泌青膿素（Pyocyanin，藍綠色高氧化還原活性毒素）和黃膿素（Pyoverdine，黃綠色熒光 siderophore 鐵載體）。

• GFP 熒光標(biāo)記特性：

  • 利用染色體定點整合（如使用 Mini-Tn7 轉(zhuǎn)座子系統(tǒng)結(jié)構(gòu)性整合至染色體單一中性位點 attTn7）或高穩(wěn)定性低拷貝質(zhì)粒載體，將綠色熒光蛋白基因（gfp，如增強型 egfp 或穩(wěn)定型 gfp-mut2）導(dǎo)入 PAO1 基因組中。

  • 激發(fā)與發(fā)射波長：該綠色熒光蛋白受強啟動子（如 tac, lac 或結(jié)構(gòu)性表達的 PclpB 啟動子）驅(qū)動?；罹谑艿讲ㄩL約為 488 nm 的藍光或紫外光激發(fā)時，會放射出波長約為 507 - 510 nm 的強勁綠色熒光。熒光強度高，抗光漂白（Photobleaching）能力強。

• 生物安全級別：2級（BSL-2）。作為臨床重要條件致病菌，活菌操作必須在二級生物安全柜內(nèi)進行。

二 核心科研價值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

PAO1-GFP 菌株將銅綠假單胞菌的強致病特征與現(xiàn)代顯微成像及流式分析技術(shù)結(jié)合，是空間組織學(xué)和單細胞行為學(xué)研究的利器：

1. 共聚焦顯微鏡下生物膜三維空間結(jié)構(gòu)重塑（3D Biofilm Architecture）：

   銅綠假單胞菌是研究生物膜（Biofilm）的國際金標(biāo)準(zhǔn)。PAO1-GFP 在流體剪切力細胞（Flow Cells）或蓋玻片表面組裝成復(fù)雜生物膜時，利用激光共聚焦掃描顯微鏡（CLSM）進行 Z 軸層切掃描，可清晰重構(gòu)出生物膜的三維“蘑菇狀”空間微結(jié)構(gòu)、水通道分布以及胞外基質(zhì)包裹形態(tài)，無需后期熒光染色破壞。

2. 多菌種交互與協(xié)同定殖可視化（Polymicrobial Interactions）：

   在口腔菌斑、慢性傷口或肺部混合感染研究中，PAO1-GFP 常與帶有紅色熒光標(biāo)記（如 mChrry/tdTomato）的其他條件致病菌（如金黃色葡萄球菌 S. aureus）共同孵育。利用多通道熒光顯微鏡，可以實時動態(tài)捕捉不同菌種在空間上的共定位、競爭性排斥、機械性纏繞以及群體感應(yīng)（QS）介導(dǎo)的協(xié)同包裹行為。

3. 宿主細胞入侵、胞內(nèi)寄生與吞噬動力學(xué)研究（Host-Pathogen Interactions）：

   常用于侵染體外培養(yǎng)的人巨噬細胞、肺泡上皮細胞（A549）或血管內(nèi)皮細胞。通過流式細胞術(shù)（FACS）可高通量定量分析巨噬細胞對 PAO1-GFP 的吞噬吞吐率；利用熒光顯微鏡可清晰定位活菌在宿主細胞質(zhì)內(nèi)的定殖增殖、逃逸溶酶體降解的軌跡。

三 實驗室菌株復(fù)蘇、擴增傳代與冷凍保存標(biāo)準(zhǔn)步驟

1. 擴增培養(yǎng)基與選擇抗性配置

PAO1-GFP 表現(xiàn)出極為強健的營養(yǎng)適應(yīng)性，但在日常培養(yǎng)中仍需注意維持其標(biāo)記的遺傳嚴(yán)謹性：

• 基礎(chǔ)培養(yǎng)基：LB（Lysogeny Broth）肉湯/固體瓊脂平板（推薦），或胰酪胨大豆（TSB/TSA）培養(yǎng)基。

• 選擇性維持抗生素（關(guān)鍵，依具體構(gòu)建而定）：為了在長期連續(xù)傳代中徹底杜絕 gfp 基因因同源重組或轉(zhuǎn)座子丟失而出現(xiàn)熒光流失，必須在常規(guī)擴增平板/肉湯中補充對應(yīng)工程載體所帶的抗生素。常見的選擇壓包括：慶大霉素（Gentamicin，染色體整合型常選用 15 - 30 ug/mL）、卡那霉素（Kanamycin，工作濃度 50 ug/mL）或四環(huán)素（Tetracycline，工作濃度 10 - 20 ug/mL）。如果是通過外源質(zhì)粒維持，抗生素必不可少；若是染色體轉(zhuǎn)座子定點整合型，短中期傳代不加抗生素亦可穩(wěn)定表達熒光。

2. 凍干粉/凍存菌種復(fù)蘇步驟

1. 將含有對應(yīng)選擇抗性的 LB 固體平板提前置于常規(guī)培養(yǎng)箱或室溫平衡，保持表面干爽。

2. 從超低溫冰箱或液氮中取出 PAO1-GFP 的凍存管，置于冰上融化，或在生物安全柜內(nèi)小心打開復(fù)蘇安瓿管。

3. 用無菌移液管吸取約 200 - 500 uL 的常規(guī)無抗 LB 肉湯加入管內(nèi)，輕柔吹打菌塊使其完全懸浮溶解。

4. 吸取全量菌懸液，傾倒或用涂布棒密集涂布于選擇性 LB 固體平板的濃集區(qū)域，隨后使用接種環(huán)進行常規(guī)四區(qū)劃線，以便挑取單菌落。

5. 將平板倒置置于 37 攝氏度普通有氧培養(yǎng)箱中，連續(xù)孵育 14 至 18 小時（過夜）。次日長出邊緣微扁平、常帶有特征性藍綠色漫延和金屬光澤的健壯菌落。

3. 日常傳代與熒光活性監(jiān)測

• 熒光檢測：將長有單菌落的平板置于手提式紫外燈或凝膠成像系統(tǒng)的藍光透射儀（Blue Light Transilluminator，約 470 nm）上，開啟激發(fā)光，肉眼即可直接觀察到大大小小的菌落爆發(fā)出極為耀眼、翠綠色的明亮綠色熒光（自發(fā)分泌的黃膿素在紫外下雖也有綠光，但 GFP 熒光具有極強的像素聚焦感且強度遠超背景）。

• 傳代擴增：挑取平板上單個綠色熒光飽滿、邊緣清晰的單菌落，接種至 5 - 10 mL 選擇性液體 LB 肉湯試管中。置于 37 攝氏度、200 - 220 rpm 振蕩搖床內(nèi)，連續(xù)培養(yǎng) 10 至 14 小時（通常過夜搖菌）。為了開展精密成像或定量流式分析，建議選用對數(shù)生長中晚期的菌體，此時細菌活性最高，GFP 蛋白折疊最充分且熒光強度與菌落數(shù)成正比。

4. 菌株長期冷凍保存

• 長期甘油冷凍法：采集處于對數(shù)生長晚期、綠色熒光強度達到峰值的液體培養(yǎng)物。

• 凍存操作：在無菌分裝管內(nèi)，將 750 uL 液體菌懸液與 250 uL 滅菌高純無菌甘油（或直接使用含 20% 甘油的專屬保菌液）輕柔混勻，使最終甘油保護劑工作濃度達到 20% 左右。

• 保存條件：混勻后，立即將凍存管直接投入 -80 攝氏度 超低溫冰箱，或者放入 液氮（-196 攝氏度） 蒸氣層內(nèi)。在此穩(wěn)定的超低溫物理狀態(tài)下，PAO1-GFP 的活菌數(shù)量及 GFP 熒光發(fā)光活性可維持數(shù)年以上不發(fā)生衰減。

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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