BioVector? pDR111_Bga no NLS 枯草芽孢桿菌異源表達(dá)與基因組整合載體 BioVector? pDR111_Bga no NLS Bacillus subtilis Heterologous Expression and Integration Vector

第一部分：中文說明

一、 產(chǎn)品基本信息與詳細(xì)特征描述

• 產(chǎn)品名稱：BioVector? pDR111_Bga no NLS 枯草芽孢桿菌異源表達(dá)與基因組整合載體

• 載體名稱：BioVector? pDR111_Bga no NLS

• 質(zhì)粒類型：枯草芽孢桿菌表達(dá)與染色體整合穿梭載體 (B. subtilis Expression & Integration Vector)

• 抗性基因：大腸桿菌選擇抗性為氨芐青霉素 (Ampicillin, 100 μg/mL)；枯草芽孢桿菌整合選擇抗性為分枝高哌素/光譜霉素 (Spectinomycin, 100 μg/mL)

• 啟動(dòng)子區(qū)域：Pspank 或 Pspank(hy) 強(qiáng)誘導(dǎo)啟動(dòng)子（IPTG 可調(diào)控誘導(dǎo)系統(tǒng)）

• 整合位點(diǎn)：amyE 位點(diǎn)（通過雙交換同源重組）

• 報(bào)告/融合標(biāo)簽：Bga（來(lái)自青霉菌或內(nèi)源的 β-半乳糖苷酶衍生標(biāo)簽），且去除了核定位信號(hào) (no NLS, No Nuclear Localization Signal)

• 生物安全級(jí)別：1級(jí) (BSL-1)

• 詳細(xì)特征描述：BioVector? pDR111_Bga no NLS 是一種專門針對(duì)枯草芽孢桿菌 (Bacillus subtilis) 開發(fā)的高級(jí)通用型整合表達(dá)載體。該質(zhì)粒的設(shè)計(jì)核心是在大腸桿菌中可作為高拷貝質(zhì)粒進(jìn)行克隆與擴(kuò)增（依靠其自帶的 bla 標(biāo)記和 pUC 復(fù)制子），但在導(dǎo)入枯草芽孢桿菌后，由于缺乏該宿主特異性的復(fù)制起點(diǎn)，其必須通過兩側(cè)的 amyE 基因同源翼（amyE-front 和 amyE-back）發(fā)生高效率的雙交換（Double-crossover）同源重組。該過程將目的表達(dá)框及光譜霉素抗性基因精準(zhǔn)嵌入宿主染色體的 amyE 位點(diǎn)，實(shí)現(xiàn)單拷貝基因組穩(wěn)定集成，杜絕了工業(yè)發(fā)酵中因質(zhì)粒丟失造成的表達(dá)波動(dòng)。該載體集成了受 LacI 嚴(yán)密壓制的 IPTG 誘導(dǎo)型強(qiáng)啟動(dòng)子，且融合了 Bga 報(bào)告基因表型標(biāo)簽，關(guān)鍵的是，該版本移除了核定位信號(hào) (no NLS)。這使得表達(dá)出的融合蛋白或報(bào)告蛋白能夠自由、均勻地分布在原核生物的細(xì)胞質(zhì)（Cytoplasm）中，而不會(huì)發(fā)生異常的亞細(xì)胞空間聚集。這一改動(dòng)極大優(yōu)化了異源蛋白在細(xì)菌胞內(nèi)的折疊動(dòng)態(tài)，避免了由于人工定位序列引起的細(xì)胞毒性，是枯草芽孢桿菌胞內(nèi)高效高質(zhì)表達(dá)、發(fā)酵酶制劑改良及精細(xì)定量轉(zhuǎn)錄分析的理想分子利器。

二、 細(xì)胞培養(yǎng)與質(zhì)粒克隆條件

• 大腸桿菌克隆與擴(kuò)增條件：進(jìn)行外源片段連接與重組構(gòu)建時(shí)，可將重組子轉(zhuǎn)化至標(biāo)準(zhǔn)大腸桿菌克隆感受態(tài)細(xì)胞（如 DH5α 或 Top10）。使用無(wú)菌配制的 BioVector? LB 液體培養(yǎng)基或 LB 固體瓊脂平板，并在其中添加終濃度為 100 微克每毫升的 BioVector? 氨芐青霉素。置于 37°C 恒溫振蕩培養(yǎng)箱中以每分鐘 200 到 220 轉(zhuǎn)的轉(zhuǎn)速孵育過夜。

• 枯草芽孢桿菌轉(zhuǎn)化與篩選條件：純化驗(yàn)證后的整合重組質(zhì)粒，通過電轉(zhuǎn)化或天然化學(xué)感受態(tài)方法導(dǎo)入枯草芽孢桿菌。轉(zhuǎn)化后的復(fù)蘇細(xì)胞需接種于添加了終濃度為 100 微克每毫升 BioVector? 光譜霉素的營(yíng)養(yǎng)瓊脂或 LB 固體平板上。培養(yǎng)箱環(huán)境參數(shù)嚴(yán)格控制在 37°C。

三、 枯草芽孢桿菌整合轉(zhuǎn)錄及表型鑒定步驟

1. 質(zhì)粒提取與線性化：使用 BioVector? 高純度無(wú)內(nèi)毒素質(zhì)粒提取試劑盒回收重組質(zhì)粒。為了大幅度提高染色體雙交換重組成功率，建議在轉(zhuǎn)化前使用限制性內(nèi)切酶切斷載體的非同源主鏈區(qū)，進(jìn)行質(zhì)粒線性化處理。

2. 感受態(tài)轉(zhuǎn)化與復(fù)蘇：將線性化 DNA 加入處于感受態(tài)生長(zhǎng)期末期的枯草芽孢桿菌中，微量混勻。在 37°C 下靜態(tài)孵育 30 到 40 分鐘使其攝取 DNA。隨后加入適量預(yù)熱的富集肉湯，置于搖床中 37°C 低速?gòu)?fù)蘇 1 小時(shí)，以使光譜霉素抗性基因充分表達(dá)。

3. 抗性篩選：將復(fù)蘇菌液均勻涂布在含有 100 微克每毫升光譜霉素的篩選平板上，置于 37°C 培養(yǎng) 18 到 24 小時(shí)，直至長(zhǎng)出清晰的光譜霉素抗性單克隆。

4. amyE 位點(diǎn)滅活與 Bga 報(bào)告驗(yàn)證：由于雙交換會(huì)導(dǎo)致宿主內(nèi)源 amyE（α-淀粉酶）基因斷裂，可挑取單克隆點(diǎn)樣于含有 1% 可溶性淀粉的瓊脂平板上，37°C 培養(yǎng)過夜，傾倒 BioVector? 碘液。若菌落周圍未形成透明水解圈（淀粉不被水解），證明成功整合于 amyE 位點(diǎn)。同時(shí)，由于融合了 Bga（β-半乳糖苷酶活性衍生片段且 no NLS），可通過在含有 X-gal 且不含 NLS 干擾的平板上觀察菌落呈均勻藍(lán)色，來(lái)定性評(píng)估轉(zhuǎn)錄活性。

四、 質(zhì)粒與工程菌株的保藏技術(shù)

• 大腸桿菌菌種凍存：將克隆有正確質(zhì)粒的大腸桿菌接種擴(kuò)增至對(duì)數(shù)生長(zhǎng)中期（OD600 達(dá)到 0.6 左右）。吸取 700 微里菌液與 300 微里無(wú)菌的 BioVector? 細(xì)胞級(jí)甘油在無(wú)菌管中徹底混勻（最終甘油濃度為 30%），直接放入 零下 80°C 超低溫冰箱長(zhǎng)期保藏。

• 純化質(zhì)粒 DNA 儲(chǔ)存：質(zhì)粒 DNA 需溶解于無(wú)菌的 BioVector? TE 緩沖液（pH 8.0）或無(wú)核酸酶的超純水中。分裝成小體系后，置于 零下 20°C 冰箱中可穩(wěn)定保存數(shù)年，避免頻繁反復(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? 出品的質(zhì)粒經(jīng)過嚴(yán)格的序列完整性及功能驗(yàn)證。通過高通量 Sanger 測(cè)序確認(rèn) Pspank 啟動(dòng)子區(qū)、LacI 結(jié)合位點(diǎn)、Bga 標(biāo)記區(qū)及獨(dú)特的 no NLS（無(wú)核定位信號(hào)） 關(guān)鍵突變區(qū)域全長(zhǎng)序列 100% 準(zhǔn)確無(wú)漂移；酶切物理電泳圖譜與設(shè)計(jì)圖譜完全相符；無(wú)宿主核酸及 DNase/RNase 酶污染。

• 核心實(shí)驗(yàn)應(yīng)用方向：該整合載體主要用于枯草芽孢桿菌內(nèi)異源重組蛋白的非聚集性、均勻胞質(zhì)表達(dá)；用于需要排除核定位信號(hào)干擾的原核細(xì)胞亞細(xì)胞定位對(duì)照實(shí)驗(yàn)；在大腸桿菌-芽孢桿菌穿梭系統(tǒng)中對(duì)復(fù)雜啟動(dòng)子和轉(zhuǎn)錄調(diào)節(jié)因子進(jìn)行體內(nèi)定量滴定；以及構(gòu)建染色體單拷貝、高度遺傳穩(wěn)定的工業(yè)級(jí)酶制劑生產(chǎn)菌株。

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector? pDR111_Bga no NLS Bacillus subtilis Heterologous Expression and Integration Vector

• Vector Name: BioVector? pDR111_Bga no NLS

• Plasmid Type: B. subtilis Expression & Chromosomal Integration Shuttle Vector

• Selection Marker: Ampicillin (100 μg/mL) for propagation in E. coli; Spectinomycin (100 μg/mL) for chromosomal integration selection in B. subtilis.

• Promoter System: Robust Pspank or Pspank(hy) IPTG-inducible promoter infrastructure.

• Integration Target: amyE locus (via double-crossover homologous recombination)

• Reporter / Fusion Tag: Bga (β-galactosidase derived marker) configured with no Nuclear Localization Signal (no NLS).

• Biosafety Level: BSL-1

• Detailed Description: BioVector? pDR111_Bga no NLS is an advanced, highly specialized expression and site-specific integration vector meticulously engineered for genetic manipulation within Bacillus subtilis. The structural design permits high-copy plasmid replication and routine cloning within standard E. coli hosts via its pUC origin and bla (Ampicillin) selection marker. Crucially, because it lacks a functional replication origin for Gram-positive hosts, the plasmid functions strictly as a suicide integration vector upon introduction into B. subtilis. Flanked by homologous segments matching the front and back regions of the native amyE gene, it drives a high-efficiency double-crossover event that seamlessly embeds the expression cassette and the companion Spectinomycin marker into the host genome. This single-copy chromosomal integration resolves segregational instability and expression leakage inherent to episomal plasmids. Controlled by a tightly repressed, IPTG-inducible promoter, the expression cassette features a Bga reporter gene deliberately engineered with no NLS. Eliminating the nuclear localization signal ensures that the synthesized fusion protein or reporter distributes evenly across the prokaryotic cytoplasm instead of forming artificial sub-cellular clusters or aggregates. This modification optimizes protein folding behavior, mitigates transport-associated toxicity, and serves as an exceptional tool for cellular tracking, promoter titration, and metabolic engineering.

II. Culture Conditions and Cloning Parameters

• E. coli Propagation and Cloning Requirements: For traditional insert ligation, sequence modification, or large-scale plasmid harvesting, the vector should be transformed into E. coli competent cells (e.g., DH5α or Top10). Propagate transformed cells using sterile BioVector? LB Liquid Medium or LB Agar plates supplemented with BioVector? Ampicillin at a final working concentration of 100 micrograms per milliliter. Incubate overnight in a temperature-controlled shaking incubator at 37°C with an agitation speed of 200 to 220 RPM.

• B. subtilis Selection and Environmental Benchmarks: Sequence-verified recombinant constructs are introduced into B. subtilis via chemical competence or electroporation protocols. Transformed populations must be selected on BioVector? Nutrient Agar or LB Agar plates containing a final concentration of 100 micrograms per milliliter of BioVector? Spectinomycin. Constantly maintain incubation parameters at 37°C.

III. Standardized B. subtilis Integration and Phenotypic Verification Protocol

1. Plasmid Harvesting and Linearization: Extract the recombinant plasmid DNA using a BioVector? High-Purity Endotoxin-Free Plasmid Extraction Kit. To significantly enhance the frequency of double-crossover homologous recombination over single-crossover events, linearize the plasmid DNA using a restriction endonuclease that cleaves uniquely within the non-homologous vector backbone prior to transformation.

2. Competent Cell Transformation and Outgrowth: Add the linearized DNA directly into a suspension of B. subtilis competent cells harvested at their optimal receptive stage. Mix gently and incubate statically at 37°C for 30 to 40 minutes to facilitate DNA uptake. Supplement the transformation mixture with pre-warmed recovery broth and agitate at low speed for 1 hour at 37°C to allow full phenotypic expression of the Spectinomycin resistance gene.

3. Selective Plate Plating: Spread the recovered outgrowth evenly onto selective plates supplemented with 100 micrograms per milliliter of Spectinomycin. Invert the plates and incubate at 37°C for 18 to 24 hours until robust single transformants materialize.

4. Amylase Disruption and Bga Reporter Validation: Successful double-crossover events disrupt the native amyE gene encoding alpha-amylase. Patch individual spectinomycin-resistant colonies onto a BioVector? Nutrient Agar plate containing 1% soluble starch and grow overnight at 37°C. Flood the surface with BioVector? Iodine Solution; colonies lacking a surrounding clear zone (starch remaining unhydrolyzed) initially confirm target integration at the amyE locus. Concurrently, the expression of the Bga tag (devoid of NLS constraints) can be evaluated qualitatively on plates supplemented with IPTG and X-gal, yielding a uniform blue colony phenotype confirming localized cytoplasmic reporter function.

IV. Plasmid Preservation and Long-Term Storage Methodology

• Host Bacterial Glycerol Stock Preparation: For long-term archiving of E. coli strains carrying the validated construct, cultivate the host culture into its active logarithmic growth phase (OD600 around 0.6). Thoroughly combine 700 microliters of the bacterial culture with 300 microliters of sterile, BioVector? Cell-Grade Glycerol to achieve a final concentration of 30% glycerol inside a sterile cryovial. Store directly inside a minus 80°C ultra-low temperature freezer.

• Purified Plasmid DNA Archiving: Store purified plasmid DNA dissolved in sterile BioVector? TE Buffer (pH 8.0) or nuclease-free ultra-pure water. Aliquot into small single-use batches and preserve at minus 20°C. Repetitive freeze-thaw cycles must be strictly avoided to prevent physical double-stranded DNA cleavage.

V. Quality Control and Research Application Guidelines

• Quality Control Standards: The BioVector? pDR111_Bga no NLS vector undergoes stringent quality validation. High-throughput Sanger sequencing guarantees 100% sequence accuracy across the Pspank promoter region, the LacI repressor binding domains, the Bga open reading frame, and the critical mutated no NLS boundary. Restriction enzyme digestion mapping confirms complete structural alignment with theoretical plasmid maps. The product is certified free from contaminating host chromosomal DNA and active nucleases (DNase/RNase).

• Core Experimental Applications: This specialized integration vector is widely applied for the non-aggregating, homogenous cytoplasmic expression of heterologous proteins in B. subtilis; as a negative control framework in sub-cellular localization studies to exclude artificial nuclear-targeting peptide signaling; for the in vivo quantitative titration and kinetic assessment of complex promoters and transcription factors; and for fabricating single-copy, genetically stable industrial production strains for high-yield enzyme manufacturing.

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