BioVector? pDG1663 枯草芽孢桿菌整合表達載體 BioVector? pDG1663 Bacillus subtilis Integration Expression Vector

第一部分：中文說明

一、 產品基本信息與詳細特征描述

• 產品名稱：BioVector? pDG1663 枯草芽孢桿菌整合表達載體

• 載體名稱：BioVector? pDG1663

• 質粒類型：枯草芽孢桿菌整合型載體 (Bacillus subtilis Integration Vector)

• 抗性基因：大腸桿菌選擇抗性為氨芐青霉素 (Ampicillin)；枯草芽孢桿菌整合選擇抗性為紅霉素 (Erythromycin) 與林可霉素 (Lincomycin) 聯合選擇（即 MLS 抗性表型）

• 整合位點：amyE 位點（雙交換同源重組）

• 生物安全級別：1級 (BSL-1)

• 詳細特征描述：BioVector? pDG1663 是一種專門用于枯草芽孢桿菌 (Bacillus subtilis) 基因組定點整合與異源基因高效表達的經典分子生物學載體。該載體設計核心包含大腸桿菌 (E. coli) 復制子，允許在通用大腸桿菌宿主中進行高效克隆、擴增與質粒提取。載體不具備在枯草芽孢桿菌中自主復制的能力，而是依賴于其兩側側翼的 amyE 基因同源片段。當轉入枯草芽孢桿菌后，載體通過雙交換（Double-crossover）同源重組事件，將目的基因片段及伴隨的 MLS 抗性標記精準插入到宿主染色體的 amyE（α-淀粉酶）位點中，導致宿主內源 amyE 基因失活。該整合策略能夠實現目的基因在宿主基因組中的單拷貝單向穩(wěn)定集成，徹底解決因質粒不穩(wěn)定或由于自發(fā)丟失導致的表達量波動問題。其自帶的強啟動子區(qū)域可驅動下游異源蛋白的高效、持續(xù)表達，是原核表達、工業(yè)酶制劑改造、代謝途徑構建及枯草芽孢桿菌基因功能研究的理想分子工具。

二、 細胞培養(yǎng)與質?？寺l件

• 大腸桿菌克隆與擴增條件：為了進行載體克隆、片段插入或質粒大量提取，應將質粒轉化至常規(guī)大腸桿菌感受態(tài)細胞（如 DH5a 或 Top10）。使用配置好的 BioVector? LB 液體培養(yǎng)基或 BioVector? LB 固體瓊脂培養(yǎng)基，并在其中添加終濃度為 100 微克每毫升的 BioVector? 氨芐青霉素。在 37°C 恒溫振蕩培養(yǎng)箱中以每分鐘 200 到 220 轉的轉速孵育過夜。

• 枯草芽孢桿菌轉化與篩選條件：完成目的片段克隆的高質量質粒，通過化學轉化或電轉化方式導入枯草芽孢桿菌感受態(tài)細胞。轉化后的細胞需要接種于添加了 MLS 選擇性抗生素的 BioVector? 營養(yǎng)瓊脂或 BioVector? 營養(yǎng)肉湯培養(yǎng)基中。標準篩選濃度通常為每毫升 1 微克的 BioVector? 紅霉素結合每毫升 25 微克的 BioVector? 林可霉素。孵育環(huán)境參數設定為 37°C 恒溫培養(yǎng)。

三、 枯草芽孢桿菌整合與傳代操作步驟

1. 質粒制備與線性化：使用 BioVector? 高純度質粒提取試劑盒從大腸桿菌中提取足量的 BioVector? pDG1663 重組質粒。為了大幅度提高枯草芽孢桿菌中的雙交換同源重組效率，建議在轉化前使用限制性內切酶在非同源區(qū)域內對質粒進行線性化處理。

2. 枯草感受態(tài)轉化：將線性化或超螺旋狀態(tài)的重組質粒加入到處于感受態(tài)生長末期的枯草芽孢桿菌懸液中，輕柔混勻，于 37°C 靜態(tài)孵育 30 到 45 分鐘以促進 DNA 分子的攝取。

3. 轉化后恢復：向轉化混合物中加入適量預熱的富集培養(yǎng)基（如 BioVector? Spizizen 最小培養(yǎng)基或預熱重組恢復肉湯），置于 37°C 振蕩培養(yǎng)箱中低速復蘇 1 到 1.5 小時，使其抗性基因得到充分表達。

4. 抗性平板篩選：將復蘇后的菌液均勻涂布于含有特定濃度 BioVector? 紅霉素與林可霉素聯合抗性的篩選平板上，倒置放入 37°C 培養(yǎng)箱中孵育 18 到 24 小時，直至平板上長出清晰的單克隆。

5. 整合表型驗證 (amyE 陰性測試)：由于雙交換整合會導致枯草芽孢桿菌的 amyE 基因斷裂，可通過淀粉水解實驗進行快速表型鑒定。將長出的轉化子挑取點樣于含有 1% 可溶性淀粉的 BioVector? 營養(yǎng)瓊脂平板上，37°C 培養(yǎng)過夜。隨后向平板上傾倒適量 BioVector? 碘液。若菌落周圍未出現透明的水解圈（即淀粉不被水解），則初步證實目的片段已成功整合于 amyE 位點。隨后應提取基因組進行 PCR 構建驗證。

四、 質粒凍存與長期保藏技術

• 大腸桿菌菌種凍存：克隆有正確質粒的大腸桿菌菌液，在對數生長旺盛期（OD600 達到 0.6 到 0.8 時），吸取 700 微里菌液與 300 微里無菌的 BioVector? 細胞級甘油（最終甘油濃度為 30%）在無菌凍存管中充分混勻。利用程序降溫或直接置于零下 80°C 超低溫冰箱中進行長期、穩(wěn)定的保藏。

• 質粒 DNA 儲存：純化后的質粒 DNA 應當溶解于無菌的 BioVector? TE 緩沖液或無酸性超純水中，測量濃度后將其分裝。置于零下 20°C 冰箱中可穩(wěn)定保存數年，避免反復凍融以防止質粒 DNA 鏈斷裂。

五、 質量控制與科研應用指南

• 質量控制標準：BioVector? pDG1663 載體經過嚴格的純度、濃度和序列完整性檢測。通過酶切電泳分析驗證其質粒圖譜的準確性；通過高通量測序確認克隆位點及關鍵抗性基因序列無突變；確保無外源核酸、核酸酶（DNase/RNase）及細菌內毒素污染。

• 主要實驗應用：該整合載體主要用于枯草芽孢桿菌異源蛋白表達系統的構建、工業(yè)生產用工程菌株的遺傳改造、穩(wěn)定多拷貝表達株系的前體構建、復雜代謝通路基因的染色體逐個敲入，以及芽孢桿菌內部調控元件與啟動子強度的體內定量分析。

PART 2: ENGLISH SECTION

I. General Information and Detailed Product Characterization

• Product Name: BioVector? pDG1663 Bacillus subtilis Integration Expression Vector

• Vector Name: BioVector? pDG1663

• Plasmid Type: Bacillus subtilis Integration Vector

• Antibiotic Resistance: Ampicillin for selection in E. coli; Macrolide-Lincosamide-Streptogramin B (MLS) resistance (Erythromycin and Lincomycin combined) for chromosomal integration selection in B. subtilis.

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

• Biosafety Level: BSL-1

• Detailed Description: BioVector? pDG1663 is a classical molecular biology vector dedicated to site-specific chromosomal integration and highly stable heterologous gene expression in Bacillus subtilis. The design of this vector incorporates an E. coli replicon, allowing for efficient cloning, modification, and high-yield plasmid extraction within common laboratory E. coli host strains. Crucially, the plasmid lacks a functional replication origin for Bacillus subtilis, meaning its persistence in the host relies entirely on homologous recombination directed by the flank sequences of the amyE gene. Upon transformation into B. subtilis, a double-crossover event integrates the target gene cassette along with the MLS resistance marker precisely into the chromosomal amyE (alpha-amylase) locus, simultaneously rendering the endogenous amylase gene inactive. This integration strategy secures a single-copy, highly stable genotypic preservation within the host genome, eliminating expression fluctuations caused by plasmid instability or segregational loss. Driven by its embedded strong promoter region, it supports sustained and efficient synthesis of target proteins, serving as an outstanding molecular tool for prokaryotic expression, industrial enzyme optimization, metabolic engineering, and functional genomics.

II. Culture Conditions and Cloning Parameters

• E. coli Maintenance and Amplification: For general cloning, insert ligation, or high-yield plasmid purification, the vector should be transformed into standard E. coli competent cells (e.g., DH5a or Top10). Cells must be cultivated using professionally prepared BioVector? LB Liquid Medium or BioVector? LB Agar Medium supplemented with BioVector? Ampicillin at a final concentration of 100 micrograms per milliliter. Incubate in a temperature-controlled shaking incubator at 37°C with an agitation speed of 200 to 220 RPM overnight.

• B. subtilis Selection and Growth Conditions: Verified recombinant plasmids carrying the target fragment are introduced into B. subtilis competent cells via chemical transformation or electroporation. Successfully transformed colonies are strictly selected on BioVector? Nutrient Agar or BioVector? Nutrient Broth containing MLS antibiotics. The standard working concentrations are 1 microgram per milliliter of BioVector? Erythromycin combined with 25 micrograms per milliliter of BioVector? Lincomycin. The incubation environment must be constantly maintained at 37°C.

III. Standardized B. subtilis Integration and Subculturing Protocol

1. Plasmid Preparation and Linearization: Extract a sufficient quantity of the recombinant BioVector? pDG1663 plasmid using a BioVector? High-Purity Plasmid DNA Extraction Kit. To maximize the frequency of double-crossover homologous recombination in Bacillus subtilis, it is highly recommended to linearize the plasmid using a restriction enzyme that cuts within a non-homologous backbone region prior to transformation.

2. Competent Cell Transformation: Add the linearized or supercoiled recombinant plasmid directly into the B. subtilis competent cell suspension harvested at the late-competent growth stage. Mix very gently and incubate statically at 37°C for 30 to 45 minutes to facilitate DNA uptake by the cells.

3. Post-Transformation Recovery: Supplement the mixture with an appropriate volume of pre-warmed recovery broth, such as BioVector? Spizizen Minimal Medium or a rich outgrowth broth. Incubate in a shaking incubator at 37°C at a low agitation speed for 1 to 1.5 hours to allow adequate phenotypic expression of the antibiotic resistance genes.

4. Antibiotic Plate Selection: Spread the recovered culture evenly onto selective plates containing the designated concentrations of combined BioVector? Erythromycin and Lincomycin. Invert the plates and incubate them in a 37°C incubator for 18 to 24 hours until distinct single colonies appear.

5. Integration Verification (amyE Negative Test): Because successful double-crossover integration disrupts the native amyE gene, a starch hydrolysis assay provides rapid phenotypic identification. Patch selected transformants onto a BioVector? Nutrient Agar plate supplemented with 1% soluble starch and grow overnight at 37°C. Flood the plate surface with BioVector? Iodine Solution. The absence of a clear halo or zone of clearing around the colony (indicating starch remains unhydrolyzed) initially confirms target integration at the amyE locus. This must be further verified by genomic DNA extraction and PCR analysis.

IV. Plasmid Preservation and Long-Term Storage Methodology

• E. coli Glycerol Stock Preservation: For long-term storage of host bacteria carrying the validated plasmid, collect E. coli cultures during their active logarithmic growth phase (OD600 between 0.6 and 0.8). Mix 700 microliters of the bacterial suspension thoroughly with 300 microliters of sterile BioVector? Cell-Grade Glycerol (achieving a final concentration of 30% glycerol) inside a sterile cryovial. Store the cryovial in a minus 80°C ultra-low temperature freezer.

• Purified Plasmid DNA Storage: Purified plasmid DNA should be dissolved in sterile BioVector? TE Buffer or nuclease-free ultra-pure water. Measure the concentration, aliquot into small volumes, and store at minus 20°C. Aliquoting prevents repetitive freeze-thaw cycles that can induce double-stranded DNA breaks.

V. Quality Control and Research Application Guidelines

• Quality Control Standards: The BioVector? pDG1663 vector undergoes stringent quality assessments regarding purity, concentration, and structural integrity. Structural alignment is confirmed via restriction enzyme digestion profiling; cloning sites and key resistance markers are fully verified via high-throughput sequencing to ensure no spontaneous mutations exist. The product is certified free from exogenous nucleic acids, host nucleases (DNase/RNase), and bacterial endotoxins.

• Core Experimental Applications: This integration vector is widely used for constructing stable heterologous protein expression systems in B. subtilis, genetic engineering of industrial production strains, fabricating stable precursor strains for multi-copy expression, chromosomal knock-in of sequential genes within complex metabolic pathways, and in vivo quantitative characterization of native regulatory elements and promoter strengths.

BioVector NTCC質粒載體菌株細胞蛋白抗體基因保藏中心

電話:400-800-2947

工作QQ/微信同號:1843439339

網址

http://www.nedfriskphoto.com