BioVector? pOST1-NubI 膜酵母雙雜交系統(tǒng)質(zhì)粒對(duì)照載體

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 質(zhì)粒名稱(chēng)：pOST1-NubI 對(duì)照質(zhì)粒（也常寫(xiě)作 pOST1-NubI，其中 NubI 的末尾為大寫(xiě)字母 I，代表異亮氨酸 Isoleucine，常被誤看作字母 "l" 或數(shù)字 "1"）。

• 分類(lèi)與應(yīng)用系統(tǒng)：屬于膜酵母雙雜交系統(tǒng)（Membrane Yeast Two-Hybrid System, 簡(jiǎn)稱(chēng) MYTH 系統(tǒng)）的核心官方陽(yáng)性對(duì)照獵物載體（Positive Control Prey Vector）。

• 核心分子構(gòu)件與遺傳學(xué)背景：

  • OST1（酵母寡糖轉(zhuǎn)移酶亞基1）：OST1 是一種定位于酵母內(nèi)質(zhì)網(wǎng)（ER）膜上的、具有高度穩(wěn)定性且非特異性結(jié)合的常駐跨膜蛋白（Integral Membrane Protein）。在此載體中作為錨定標(biāo)簽。

  • 野生型 NubI（野生型泛素 N 端）：融合在 OST1 的 C 端。泛素的 N 端片段（Nub）在第 13 位保持其原始的異亮氨酸（Isoleucine, I）。野生型 NubI 對(duì)泛素的 C 端片段（Cub）具有極其強(qiáng)烈的自發(fā)親和力（親和力常數(shù)極高）。

• 工作原理（為什么是陽(yáng)性對(duì)照？）：在膜雙雜交（MYTH）實(shí)驗(yàn)中，無(wú)論你的誘餌蛋白（Bait-Cub-TF）與 OST1 是否存在真正的生理學(xué)相互作用，只要將 pOST1-NubI 共同轉(zhuǎn)入酵母中，由于 NubI 與 Cub 之間極強(qiáng)的自發(fā)強(qiáng)親和力，它們都會(huì)在膜表面強(qiáng)行自發(fā)復(fù)成完整的偽泛素（Pseudo-ubiquitin）。這會(huì)激活胞質(zhì)內(nèi)的去泛素化酶（DUBs），切下人工轉(zhuǎn)錄因子（TF），進(jìn)而強(qiáng)行激活酵母報(bào)告基因（如 HIS3, ADE2, lacZ）。因此，該質(zhì)粒用于檢測(cè)整個(gè)系統(tǒng)的活性以及誘餌蛋白是否在細(xì)胞膜表面正確表達(dá)和定位。

• 復(fù)制子與篩選標(biāo)記：

  • 酵母復(fù)制子與選擇標(biāo)記：帶有多克隆位點(diǎn)附近的酵母選擇標(biāo)記（通常為 LEU2，用于在亮氨酸缺陷型培養(yǎng)基上篩選）。

  • 大腸桿菌復(fù)制子與抗性：含有 pUC 復(fù)制子，氨芐青霉素抗性（Ampicillin, AmpR），用于大腸桿菌中的擴(kuò)增與克隆維持。

二 核心科研價(jià)值與膜雙雜交系統(tǒng)應(yīng)用

pOST1-NubI 是開(kāi)展任何膜蛋白質(zhì)相互作用（PPI）篩庫(kù)或驗(yàn)證實(shí)驗(yàn)時(shí)不可或缺的控制基準(zhǔn)：

1. 誘餌蛋白（Bait）功能性功能驗(yàn)證：新構(gòu)建的膜蛋白誘餌載體（Bait-Cub-TF）在轉(zhuǎn)化酵母后，必須首先與 pOST1-NubI 共同轉(zhuǎn)化。若酵母在缺陷型平板上能夠良好生長(zhǎng)（顯藍(lán)色/產(chǎn)生報(bào)告反應(yīng)），則證明誘餌蛋白的 C 端（Cub-TF）已成功組裝并面向細(xì)胞質(zhì)一側(cè)，且處于可被切開(kāi)的正確拓?fù)浣Y(jié)構(gòu)中。

2. 排除偽陰性（False Negatives Trap）：如果實(shí)驗(yàn)組（誘餌 + 真實(shí)靶蛋白NubG）沒(méi)有任何信號(hào)，而 pOST1-NubI 陽(yáng)性對(duì)照組也沒(méi)有信號(hào)，說(shuō)明整個(gè) yeast 雙雜交體系（或變性切開(kāi)過(guò)程）出了故障，而非靶蛋白不結(jié)合，從而避免了科研誤判。

3. 系統(tǒng)靈敏度校準(zhǔn)：通過(guò)與 pOST1-NubG（突變體，作為陰性對(duì)照）并列平行實(shí)驗(yàn)，可清晰校準(zhǔn)出當(dāng)前測(cè)試酵母感受態(tài)菌株（如 NMY51）的背景噪音與篩選嚴(yán)謹(jǐn)度（Stringency）。

三 實(shí)驗(yàn)室質(zhì)粒復(fù)蘇、轉(zhuǎn)化、擴(kuò)增與保存標(biāo)準(zhǔn)步驟

1. 菌株與擴(kuò)增培養(yǎng)基配置

• 大腸桿菌宿主：推薦使用常規(guī)克隆菌株，如 DH5a、TOP10 或 XL10-Gold。

• 大腸桿菌培養(yǎng)體系：LB 肉湯/固體瓊脂培養(yǎng)基，添加最終工作濃度為 100 ug/mL 的氨芐青霉素（Ampicillin）。

• 酵母宿主與篩選體系：通常使用 NMY51 報(bào)告株，轉(zhuǎn)化后的篩選體系為 SD-Leu 或者是 SD-Trp-Leu（雙轉(zhuǎn)）合成缺陷型培養(yǎng)基。

2. 質(zhì)粒干粉/轉(zhuǎn)化復(fù)蘇步驟（Transformation & Recovery）

1. 若收到的是離心管內(nèi)的質(zhì)粒 DNA 干粉（或紙片），加入 20-50 uL 無(wú)菌去離子水或 TE 緩沖液，輕微震蕩并靜置 5 分鐘，使其完全溶解。

2. 取出 50-100 uL 大腸桿菌 DH5a 感受態(tài)細(xì)胞置于冰上融化。

3. 加入 1-2 uL 溶解后的 pOST1-NubI 質(zhì)粒 DNA，輕彈管底混勻，冰浴 30 分鐘。

4. 將離心管置于 42 攝氏度水浴中精確熱擊 45-60 秒，隨后立即置于冰上迅速冷卻 2 分鐘（切勿劇烈搖晃）。

5. 向管內(nèi)加入 500 uL 不含抗生素的無(wú)菌 LB 肉湯（或 SOC 培養(yǎng)基），置于 37 攝氏度振蕩孵育箱內(nèi)，以 200 rpm 復(fù)蘇勻速搖菌 45-60 分鐘。

6. 以 4000 rpm 離心 3 分鐘棄去部分上清，留約 100 uL 液體將菌體吹勻，涂布于含 100 ug/mL 氨芐青霉素的 LB 固體平板上。

7. 置于 37 攝氏度培養(yǎng)箱中倒置培養(yǎng) 14-16 小時(shí)，等待單菌落長(zhǎng)出。

3. 質(zhì)粒擴(kuò)增與高純度提取（Amplification & Extraction）

1. 從長(zhǎng)有單菌落的氨芐平板上，挑選一個(gè)邊緣清晰的健壯菌落，接種至 5-10 mL 含氨芐青霉素的 LB 液體肉湯試管中。

2. 置于 37 攝氏度、220 rpm 振蕩培養(yǎng) 12 至 16 小時(shí)（過(guò)夜培養(yǎng)） 至對(duì)數(shù)生長(zhǎng)晚期。

3. 利用標(biāo)準(zhǔn)的高純度質(zhì)粒提取試劑盒（Plasmid Miniprep Kit，基于硅膠柱離心法）收集菌體并提取質(zhì)粒 DNA。用無(wú)菌無(wú)酸的滅菌水（或堿性 Elution Buffer）洗脫，利用分光光度計(jì)測(cè)量并記錄濃度（A260/A280 比例應(yīng)在 1.8-2.0 之間）。

4. 質(zhì)粒及工程菌長(zhǎng)期保存（Storage Options）

• 質(zhì)粒 DNA 保存：提取出的純化 pOST1-NubI 質(zhì)?？煞盅b為小管，置于 -20 攝氏度 冰箱可穩(wěn)定保存 1-2 年；若置于 -80 攝氏度 超低溫冰箱中則可近乎無(wú)限期穩(wěn)定保存。避免反復(fù)凍融。

• 大腸桿菌甘油菌保存：取擴(kuò)增旺盛未完全衰亡的過(guò)夜菌液 800 uL，加入 200 uL 滅菌高純無(wú)菌甘油（最終甘油濃度約 20%），在凍存管中徹底顛倒混勻，立即投入 -80 攝氏度 超低溫冰箱內(nèi)，可連續(xù)存活數(shù)年以上。

Part 2 English Section

I General Information and Genetic Architecture

• Plasmid Designation: pOST1-NubI Control Plasmid Vector (The trailing character is the uppercase letter I, shorthand for Isoleucine, frequently misread as a lowercase "l" or numeral "1").

• System Category: Deployed as the foundational Positive Control Prey Vector inside the standard Membrane Yeast Two-Hybrid (MYTH) Assay Platforms.

• Core Genetic Architecture and Engineering Modules:

  • OST1 Anchor (Yeast Oligosaccharyltransferase Subunit 1): OST1 is a highly characterized, stable, non-specifically associating resident integral membrane protein localized to the yeast Endoplasmic Reticulum (ER) lipid bilayer. It functions as a rigid hydrophobic targeting scaffold.

  • Wild-type NubI Module (N-terminal Ubiquitin Variant): Fused downstream to the C-terminus of the OST1 anchor. The N-terminal fragment of yeast ubiquitin (Nub) preserves its evolutionary native wild-type Isoleucine residue at amino acid position 13 (Ile-13 / I).

• Mechanistic Workflow (Why It Manifests as a Positive Control): In the standard split-ubiquitin MYTH setup, regardless of whether your target membrane Bait (Bait-Cub-TF) possesses a true physiological affinity for the OST1 protein, co-transforming pOST1-NubI bypasses this requirement. Due to the exceptionally high spontaneous biochemical affinity engineered between wild-type NubI and Cub, these fragments automatically snap together upon proximity at the membrane surface. This reconstitutes a functional pseudo-ubiquitin structure that is instantly processed by local cytosolic deubiquitinating enzymes (DUBs). The synthetic transcription factor (TF) is proteolytically cleaved, translocates to the nucleus, and drives constitutive activation of yeast reporter cassettes (HIS3, ADE2, lacZ).

• Replicon Matrix and Selection Markers:

  • Yeast Vector Core: Contains a standard yeast selector marker (typically LEU2, allowing for metabolic screening on synthetic dropout medium lacking Leucine).

  • Bacterial Propagation Core: Armed with a robust high-copy pUC origin of replication paired with an Ampicillin resistance cassette (AmpR) for selection in E. coli.

II Strategic Research Value and Application Parameters

The pOST1-NubI vector is an absolute metric imperative for calibrating and establishing screening baselines across any membrane protein-protein interaction (PPI) library screening or bait validation pipeline:

1. Bait Functional Topography Profiling: Upon cloning a novel integral membrane protein into a Bait architecture (Bait-Cub-TF), it must be systematically co-transformed with pOST1-NubI. Robust growth of yeast colonies on selective deficient plates validates that the Bait expression kinetics are intact, the C-terminal Cub-TF tag is successfully routed to face the cytoplasm, and it remains sterically available for enzymatic cleavage.

2. Elimination of False Negative Blindspots: If a specific screen with a target prey array (Prey-NubG) yields zero survival clones, but the parallel pOST1-NubI positive channel also registers zero growth, this warns the investigator that the basic split-ubiquitin cleavage cascade or yeast viability has broken down, preventing misinterpretation of experimental data.

3. Assay Sensitivity and Background Calibration: Running pOST1-NubI side-by-side with pOST1-NubG (the mutated negative control counter-part containing an Ile13Gly switch) allows for precise quantification of the signal-to-noise ratio and baseline leakiness of the yeast reporter strain (e.g., NMY51).

III Thawing, Transformation, Amplification, and Storage Routines

1. Bacterial Strains and Media Configurations

• E. coli Propagation Host: Standard laboratory cloning lineages including DH5a, TOP10, or XL10-Gold competent cells.

• Bacterial Selective Medium: Lysogeny Broth (LB) liquid formula or solid agar matrices supplemented with a definitive work concentration of 100 ug/mL Ampicillin.

• Yeast Selective Medium: Standard yeast reporter host NMY51 cultivated on Synthetic Defined (SD) minimal media, specifically selective SD-Leu or double-selective SD-Trp-Leu matrices depending on co-transformation configurations.

2. Competent E. coli Transformation Routine

1. If the vector arrives as a lyophilized DNA pellet or impregnated on filter paper, resuspend the matrix in 20–50 uL of sterile molecular-grade deionized H2O or TE buffer. Allow to sit undisturbed for 5 minutes to guarantee complete solvation.

2. Thaw an aliquot of 50–100 uL competent DH5a cells gently on a chilled ice bed.

3. Deliver 1–2 uL of the solvated pOST1-NubI plasmid DNA directly into the cell suspension. Mix by very gentle flicking (do not vortex) and incubate on ice for 30 minutes.

4. Transfer the tube into a calibrated water bath set precisely at 42 degrees Celsius for a rigorous heat-shock window of 45–60 seconds. Instantly plunge the tube back into the ice bed for 2 minutes.

5. Inoculate the shocked cells with 500 uL of sterile, antibiotic-free LB broth or SOC recovery medium. Incubate the culture horizontally in a shaking incubator at 37 degrees Celsius running at 200 rpm for 45–60 minutes of out-growth recovery.

6. Concentrate the cells via a quick centrifugation run at 4000 rpm for 3 minutes. Decant the excess supernatant fluid, resuspend the pellet in the remaining ~100 uL volume, and spread evenly onto the pre-warmed selective Ampicillin-fortified LB agar plates.

7. Incubate inverted at 37 degrees Celsius for 14–16 hours until distinct colonies materialize.

3. Plasmid Amplification and Purification Downstream

1. Select a singular, well-isolated colony from the Ampicillin agar plate using a sterile pipette tip and drop it into 5–10 mL of fresh selective liquid LB broth.

2. Proliferate the culture overnight in a shaking incubator set to 37 degrees Celsius running at 220 rpm for 12–16 hours to capture cells in late-log phase density.

3. Harvest the bacterial mass and execute standard silica-column centrifugal plasmid extraction (utilizing a commercial Plasmid Miniprep Kit). Elute the bound DNA matrix using sterile DNA-free, nuclease-free water or slightly basic elution buffer. Verify the target yield and purity metrics via spectrophotometric analysis (the A260/A280 ratio must align tightly between 1.8 and 2.0).

4. Indefinite Storage Protocols

• Purified Plasmid Retention: Aliquot the recovered pOST1-NubI plasmid DNA into microfuge tubes. Storing at -20 degrees Celsius preserves functional fidelity for 1–2 years; storing at -80 degrees Celsius protects the polymer backbone against degradation matrices near-indefinitely. Restrict freeze-thaw cycles.

• Glycerol Stock Stabilization: Blend 800 uL of active, mid-log phase bacterial culture with 200 uL of sterile analytical-grade high-purity glycerol (yielding a final 20% glycerol freezing matrix) inside a cryovial. Vortex briefly to unify, and store immediately within an ultra-low -80 degrees Celsius freezer for stable maintenance stretching across years.

BioVector NTCC質(zhì)粒載體菌株細(xì)胞蛋白抗體基因保藏中心

電話:400-800-2947

工作QQ/微信同號(hào):1843439339

網(wǎng)址http://www.nedfriskphoto.com