BioVector? pPG612.1-mCherry 乳酸菌紅色熒光報(bào)告表達(dá)載體 BioVector? pPG612.1-mCherry Lactic Acid Bacteria Fluorescent Reporter Vector

第一部分：中文說(shuō)明

一、 產(chǎn)品基本信息與詳細(xì)特征描述

• 產(chǎn)品名稱(chēng)：BioVector? pPG612.1-mCherry 乳酸菌紅色熒光報(bào)告表達(dá)載體

• 載體名稱(chēng)：pPG612.1-mCherry

• 質(zhì)粒類(lèi)型：乳酸菌-大腸桿菌廣宿主游離型/整合型熒光報(bào)告穿梭載體

• 抗性基因：大腸桿菌選擇抗性為氨芐青霉素 (Ampicillin, 100 μg/mL) 或紅霉素；乳酸菌選擇抗性為紅霉素 (Erythromycin, 5–10 μg/mL) 或氯霉素（根據(jù)具體骨架衍生物而定，通常以紅霉素 ermR 為主）

• 報(bào)告基團(tuán)：mCherry 單體紅色熒光蛋白 (Monomeric Red Fluorescent Protein)

• 復(fù)制子：大腸桿菌復(fù)制子 (pUC ori) + 乳酸菌廣宿主低/高拷貝復(fù)制子（如 pSH71 或 pAMβ1 來(lái)源復(fù)制起點(diǎn)）

• 生物安全級(jí)別：1級(jí) (BSL-1)，適合食品級(jí)或常規(guī)實(shí)驗(yàn)室改造使用

• 詳細(xì)特征描述：BioVector? pPG612.1-mCherry 是一種專(zhuān)為食品級(jí)益生菌及乳酸菌屬（包括 Lactococcus、Lactobacillus、Leuconostoc 等）設(shè)計(jì)的經(jīng)典熒光報(bào)告與基因表達(dá)穿梭載體。該載體的核心是在廣宿主原核表達(dá)骨架中，下游融合了經(jīng)過(guò)密碼子優(yōu)化的 mCherry 單體紅色熒光蛋白基因。mCherry 具有極為優(yōu)異的光穩(wěn)定性、快速成熟能力以及極低的細(xì)胞毒性。其最大激發(fā)波長(zhǎng)為 587 nm，最大發(fā)射波長(zhǎng)為 610 nm，可發(fā)出明亮的紅色熒光。這不僅能完美避開(kāi)乳酸菌培養(yǎng)基（如 MRS）及菌體自身產(chǎn)生的強(qiáng)大黃色/綠色自發(fā)熒光干擾，還非常適合與經(jīng)典的 GFP 綠色熒光系統(tǒng)配合進(jìn)行雙色標(biāo)記實(shí)驗(yàn)。研究者通?？蓪⑷樗峋鷥?nèi)源的結(jié)構(gòu)性啟動(dòng)子（如 P32、P45 啟動(dòng)子）或誘導(dǎo)型啟動(dòng)子（如 Nisin 誘導(dǎo)的 PnisA 啟動(dòng)子系統(tǒng)）克隆至 mCherry 上游的多克隆位點(diǎn)（MCS），利用熒光信號(hào)實(shí)時(shí)監(jiān)測(cè)基因轉(zhuǎn)錄活性，或進(jìn)行目標(biāo)蛋白的 C 端/N 端熒光融合表達(dá)示蹤。

二、 細(xì)胞培養(yǎng)與質(zhì)?？寺l件

• 大腸桿菌克隆與擴(kuò)增條件：進(jìn)行重組子構(gòu)建、外源啟動(dòng)子連接或質(zhì)粒大量提取時(shí)，將質(zhì)粒轉(zhuǎn)化至常規(guī)大腸桿菌感受態(tài)細(xì)胞（如 DH5α 或 Top10）。使用標(biāo)準(zhǔn) BioVector? LB 液體培養(yǎng)基或固體平板，添加相應(yīng)抗生素（如 100 微克每毫升的氨芐青霉素或 150 微克每毫升的紅霉素）。于 37°C 恒溫振蕩培養(yǎng)箱中以每分鐘 200 轉(zhuǎn)的轉(zhuǎn)速孵育過(guò)夜。

• 乳酸菌轉(zhuǎn)化與篩選條件：純化后的重組質(zhì)粒通過(guò)高壓電轉(zhuǎn)化法導(dǎo)入乳酸菌（如 L. lactis 或 L. plantarum）的高滲電轉(zhuǎn)感受態(tài)細(xì)胞中。轉(zhuǎn)化后的細(xì)胞需在 GM17 或 MRS 液體培養(yǎng)基中于 30°C 靜態(tài)復(fù)蘇 1.5 到 2 小時(shí)。隨后涂布于含有 5 至 10 微克每毫升 BioVector? 紅霉素的 GM17/MRS 固體瓊脂平板上。乳酸菌的培養(yǎng)通常在 30°C 恒溫培養(yǎng)箱中進(jìn)行靜態(tài)（厭氧或微需氧）孵育 24 至 48 小時(shí)。

三、 熒光監(jiān)測(cè)與胞內(nèi)影像學(xué)觀察操作步驟

1. 種子液制備：挑取經(jīng)測(cè)序驗(yàn)證正確的乳酸菌重組單菌落，接種于含有 5 微克每毫升紅霉素的 MRS/GM17 液體培養(yǎng)基中，30°C 靜態(tài)培養(yǎng)過(guò)夜。

2. 啟動(dòng)子激活與表達(dá)誘導(dǎo)：將過(guò)夜菌按 1:50 轉(zhuǎn)接至新鮮培養(yǎng)基中。若使用 Nisin 誘導(dǎo)型系統(tǒng)（如 NICE 系統(tǒng)），在菌體生長(zhǎng)至對(duì)數(shù)中期（OD600 約為 0.4–0.6）時(shí)，向體系中加入微量（如 1–5 ng/mL）的 BioVector? Nisin（乳鏈菌肽）開(kāi)啟轉(zhuǎn)錄；若是組成型啟動(dòng)子，則直接定期取樣。

3. 熒光定量分析：吸取 200 微里不同生長(zhǎng)階段的菌液至黑壁無(wú)底 96 孔板中，利用多功能酶標(biāo)儀進(jìn)行檢測(cè)（激發(fā)光 587 nm，發(fā)射光 610 nm）。計(jì)算“熒光強(qiáng)度/OD600”的比值，以精確評(píng)估乳酸菌在特定環(huán)境（如模擬胃腸道酸堿應(yīng)激、膽鹽壓力）下的啟動(dòng)子轉(zhuǎn)錄動(dòng)力學(xué)。

4. 顯微成像觀察：離心收集 1 毫升菌體沉淀，用無(wú)菌 PBS 緩沖液洗滌 2 次以徹底去除培養(yǎng)基成分引起的熒光背景。將菌懸液滴加于制備好的 1% 瓊脂糖薄層載玻片上，在激光共聚焦顯微鏡或高級(jí)熒光顯微鏡下（使用 mCherry 或 TxRed 濾光片通道）觀察紅色熒光。這能清晰展示異源蛋白在乳酸菌胞內(nèi)的局域化動(dòng)態(tài)，或定性鑒定重組菌的標(biāo)記效率。

四、 質(zhì)粒與乳酸菌工程菌株的保藏技術(shù)

• 乳酸菌重組菌株凍存：將處于對(duì)數(shù)生長(zhǎng)旺盛期的重組乳酸菌菌液，按照 7 比 3 的體積比與無(wú)菌的 BioVector? 細(xì)胞級(jí)甘油充分混合（最終甘油濃度為 30%）。混勻后立即分裝于凍存管中，直接置于零下 80°C 超低溫冰箱中冷凍保存。乳酸菌在 30% 甘油保護(hù)下可穩(wěn)定存活數(shù)年以上。

• 質(zhì)粒 DNA 長(zhǎng)期儲(chǔ)存：使用質(zhì)粒提取試劑盒（由于乳酸菌細(xì)胞壁極厚，從乳酸菌提取質(zhì)粒時(shí)需提前使用溶菌酶進(jìn)行破壁處理；常規(guī)克隆建議直接從大腸桿菌擴(kuò)增純化）。將純化后的質(zhì)粒溶解于 BioVector? TE 緩沖液（pH 8.0）中，分裝后置于零下 20°C 冰箱中冷凍保存，嚴(yán)禁反復(fù)凍融。

五、 質(zhì)量控制與科研應(yīng)用指南

• 質(zhì)量控制標(biāo)準(zhǔn)：BioVector? pPG612.1-mCherry 載體經(jīng)過(guò)嚴(yán)格的測(cè)序與功能性驗(yàn)證。通過(guò)全長(zhǎng)高通量測(cè)序確認(rèn) mCherry 編碼區(qū)、乳酸菌復(fù)制子及抗性選擇標(biāo)記序列 100% 正確；經(jīng)檢測(cè)不含外源核酸酶（DNase/RNase）污染。

• 核心實(shí)驗(yàn)應(yīng)用方向：

  • 啟動(dòng)子探針表征：用于篩選和定量分析乳酸菌源的高強(qiáng)度結(jié)構(gòu)性啟動(dòng)子或環(huán)境應(yīng)激響應(yīng)啟動(dòng)子。

  • 活體功能示蹤：構(gòu)建帶有紅色熒光標(biāo)記的益生菌株，用于小鼠等動(dòng)物胃腸道定殖動(dòng)態(tài)、體內(nèi)分布及定居壽命的活體成像（IVIS）與追蹤。

  • 蛋白定位與多色成像：用于異源表達(dá)蛋白在乳酸菌細(xì)胞壁錨定、胞質(zhì)定位或分泌途徑的影像學(xué)示蹤，可與表達(dá) GFP 的載體聯(lián)用。

  • 食品級(jí)發(fā)酵監(jiān)控：在乳酸發(fā)酵、干酪成熟等工藝中，利用熒光信號(hào)無(wú)損、實(shí)時(shí)地監(jiān)控益生菌的代謝與基因表達(dá)豐度。

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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