BioVector? Pseudomonas aeruginosa PAO1-lux Bioluminescent Stable Strain / PAO1-lux 發(fā)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-lux 穩(wěn)定生物發(fā)光標(biāo)記菌株。

• 物種分類：細(xì)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(rèn)、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(yùn)動(dòng)性桿菌。在常規(guī)有氧平板上生長(zhǎng)旺盛，常自發(fā)分泌青膿素（Pyocyanin，藍(lán)綠色高氧化還原活性毒素）和黃膿素（Pyoverdine，熒光 siderophore 鐵載體）。

• lux 發(fā)光標(biāo)記特性：

  • 利用染色體定點(diǎn)整合（如使用迷你轉(zhuǎn)座子 Mini-Tn5 系統(tǒng)）或高穩(wěn)定性低拷貝質(zhì)粒載體，將來源于發(fā)光桿菌（Photorhabdus luminescens）的完整的自發(fā)光 luxCDABE 操縱子結(jié)構(gòu)性導(dǎo)入 PAO1 基因組中。

  • 無需添加底物：該操縱子同時(shí)包含了負(fù)責(zé)產(chǎn)生發(fā)光底物（長(zhǎng)鏈脂肪醛）的合成酶基因（luxCDE）和負(fù)責(zé)催化發(fā)光反應(yīng)的熒光酶基因（luxAB）。因此，活菌在進(jìn)行正常的細(xì)胞能量代謝時(shí)，即可自發(fā)產(chǎn)生波長(zhǎng)約為 490 nm 的持續(xù)藍(lán)綠色生物發(fā)光（Bioluminescence），其發(fā)光強(qiáng)度與體內(nèi)的活菌數(shù)量、代謝活躍度呈嚴(yán)格的線性正相關(guān)。

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

二 核心科研價(jià)值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

PAO1-lux 菌株將銅綠假單胞菌的強(qiáng)致病特征與實(shí)時(shí)無創(chuàng)體外/體內(nèi)成像技術(shù)結(jié)合，極大簡(jiǎn)化了微生物學(xué)傳統(tǒng)終點(diǎn)檢測(cè)：

1. 體外生物膜發(fā)展動(dòng)態(tài)監(jiān)測(cè)（Biofilm Dynamic Imaging）：

   銅綠假單胞菌是研究生物膜（Biofilm）的標(biāo)桿模型。PAO1-lux 在 96 孔板或流體微流控芯片內(nèi)組裝成堅(jiān)固的生物膜時(shí)，利用高敏感性冷 CCD 倒置光學(xué)顯微鏡或多功能微孔板讀數(shù)儀，可在完全不破壞生物膜完整性、無需添加任何顯色染料的前提下，連續(xù)數(shù)十小時(shí)在線追蹤生物膜自組裝、成熟及對(duì)藥物抵抗的實(shí)時(shí)發(fā)光動(dòng)力學(xué)曲線。

2. 小動(dòng)物體內(nèi)活體感染示蹤（In Vivo Small Animal Imaging）：

   在構(gòu)建小鼠急性肺炎（Pneumonia）、燒傷創(chuàng)面感染（Burn wound infection）、角膜炎以及角膜慢性潰瘍等動(dòng)物模型時(shí)，通過活體生物成像系統(tǒng)（IVIS），科研人員可在不同時(shí)間點(diǎn)直接穿透小鼠皮膚或組織，無創(chuàng)、定量地讀取局部菌群的發(fā)光相對(duì)強(qiáng)度（RLU），繪制體內(nèi)活菌空間分布與定殖消長(zhǎng)動(dòng)力學(xué)圖譜。這避免了在每個(gè)時(shí)間點(diǎn)處死小鼠并勻漿計(jì)數(shù)菌落（CFU）的傳統(tǒng)破壞性流程。

3. 新型抗菌藥物與反生物膜策略高通量篩選（HTS for Antimicrobials）：

   可直接用于各種全自動(dòng)機(jī)器人平臺(tái)，開展小分子抗生素、天然中藥提取物、抗菌肽（AMPs）以及群體感應(yīng)（Quorum Sensing, QS）抑制劑的高通量藥敏篩選。發(fā)光值（RLU）的陡降可在一分鐘內(nèi)敏銳反映出藥物對(duì)細(xì)菌細(xì)胞壁完整性或代謝能（ATP 生成）的毀滅性破壞，極其適合抗生素殺菌動(dòng)力學(xué)曲線（Time-kill Curves）的高頻采樣。

三 實(shí)驗(yàn)室菌株復(fù)蘇、擴(kuò)增傳代與冷凍保存標(biāo)準(zhǔn)步驟

1. 擴(kuò)增培養(yǎng)基與選擇抗性配置

PAO1-lux 表現(xiàn)出極為強(qiáng)健的營(yíng)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(jǐn)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（可選）：為了在長(zhǎng)期連續(xù)傳代中徹底杜絕 lux 操縱子因基因同源重組或轉(zhuǎn)座子丟失而出現(xiàn)“發(fā)光沉默”或質(zhì)粒流失，建議在常規(guī)擴(kuò)增平板/肉湯中補(bǔ)充對(duì)應(yīng)工程載體所帶的抗生素。常見的選擇壓包括：卡那霉素（Kanamycin，工作濃度 50 ug/mL）、慶大霉素（Gentamicin，工作濃度 15 - 30 ug/mL）或四環(huán)素（Tetracycline，工作濃度 10 - 20 ug/mL），具體需根據(jù)具體構(gòu)建批次的抗性標(biāo)簽添加。若僅進(jìn)行一兩代內(nèi)的普通功能性實(shí)驗(yàn)或動(dòng)物接種，可直接使用不加抗生素的 LB 培養(yǎng)基。

2. 凍干粉/凍存菌種復(fù)蘇步驟

1. 將不含（或含對(duì)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-lux 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 LB 固體平板的濃集區(qū)域，隨后使用接種環(huán)進(jìn)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(shí)（過夜）。銅綠假單胞菌生長(zhǎng)極快，次日即可長(zhǎng)出直徑 1 - 2 mm、邊緣微扁平、常帶有特征性藍(lán)綠色漫延和金屬光澤的健壯菌落。

3. 日常傳代與發(fā)光活性監(jiān)測(cè)

• 發(fā)光檢測(cè)：將長(zhǎng)有菌落的平板或裝有搖菌液的離心管直接置于全黑暗室中，肉眼由于生理極限很難直接看到（需極暗適應(yīng)數(shù)分鐘，且強(qiáng)度取決于構(gòu)建啟動(dòng)子），建議使用微孔板發(fā)光儀、酶標(biāo)儀的 Luminescence 通道，或利用實(shí)驗(yàn)室常見的凝膠成像系統(tǒng)（關(guān)閉激發(fā)光源，僅開啟化學(xué)發(fā)光/Chemiluminescence 拍攝模式），即可捕捉到極為明亮、輪廓清晰的白色/淺藍(lán)色光斑。

• 傳代擴(kuò)增：挑取平板上單個(gè)健壯發(fā)光的單菌落，接種至 5 - 10 mL 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) 6 至 12 小時(shí)（對(duì)數(shù)生長(zhǎng)旺盛期）。由于該菌代謝劇烈，若培養(yǎng)超過 18 小時(shí)進(jìn)入平臺(tái)期晚期或衰亡期，其發(fā)光強(qiáng)度（RLU/CFU 比值）會(huì)因細(xì)胞內(nèi) ATP 庫的枯竭而大幅下滑。因此，用于體內(nèi)實(shí)驗(yàn)接種或藥物篩查的菌液，必須使用對(duì)數(shù)生長(zhǎng)期的鮮活菌體。

4. 菌株長(zhǎng)期冷凍保存

• 長(zhǎng)期甘油冷凍法：采集處于對(duì)數(shù)生長(zhǎng)中晚期（振蕩培養(yǎng)約 8 - 10 小時(shí)，OD600 約在 0.6 - 1.0 之間）、自發(fā)光值達(dá)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(hù)劑工作濃度達(dá)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-lux 的活菌數(shù)量及 lux 操縱子的發(fā)光動(dòng)力學(xué)活性可維持?jǐn)?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-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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