BioVector® pOST1-NubI 膜酵母双杂交系统质粒对照载体

一 产品基本信息与遗传学背景

• 质粒名称：pOST1-NubI 对照质粒（也常写作 pOST1-NubI，其中 NubI 的末尾为大写字母 I，代表异亮氨酸 Isoleucine，常被误看作字母 "l" 或数字 "1"）。

• 分类与应用系统：属于膜酵母双杂交系统（Membrane Yeast Two-Hybrid System, 简称 MYTH 系统）的核心官方阳性对照猎物载体（Positive Control Prey Vector）。

• 核心分子构件与遗传学背景：

  • OST1（酵母寡糖转移酶亚基1）：OST1 是一种定位于酵母内质网（ER）膜上的、具有高度稳定性且非特异性结合的常驻跨膜蛋白（Integral Membrane Protein）。在此载体中作为锚定标签。

  • 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 位保持其原始的异亮氨酸（Isoleucine, I）。野生型 NubI 对泛素的 C 端片段（Cub）具有极其强烈的自发亲和力（亲和力常数极高）。

• 工作原理（为什么是阳性对照？）：在膜双杂交（MYTH）实验中，无论你的诱饵蛋白（Bait-Cub-TF）与 OST1 是否存在真正的生理学相互作用，只要将 pOST1-NubI 共同转入酵母中，由于 NubI 与 Cub 之间极强的自发强亲和力，它们都会在膜表面强行自发复成完整的伪泛素（Pseudo-ubiquitin）。这会激活胞质内的去泛素化酶（DUBs），切下人工转录因子（TF），进而强行激活酵母报告基因（如 HIS3, ADE2, lacZ）。因此，该质粒用于检测整个系统的活性以及诱饵蛋白是否在细胞膜表面正确表达和定位。

• 复制子与筛选标记：

  • 酵母复制子与选择标记：带有多克隆位点附近的酵母选择标记（通常为 LEU2，用于在亮氨酸缺陷型培养基上筛选）。

  • 大肠杆菌复制子与抗性：含有 pUC 复制子，氨苄青霉素抗性（Ampicillin, AmpR），用于大肠杆菌中的扩增与克隆维持。

二 核心科研价值与膜双杂交系统应用

pOST1-NubI 是开展任何膜蛋白质相互作用（PPI）筛库或验证实验时不可或缺的控制基准：

1. 诱饵蛋白（Bait）功能性功能验证：新构建的膜蛋白诱饵载体（Bait-Cub-TF）在转化酵母后，必须首先与 pOST1-NubI 共同转化。若酵母在缺陷型平板上能够良好生长（显蓝色/产生报告反应），则证明诱饵蛋白的 C 端（Cub-TF）已成功组装并面向细胞质一侧，且处于可被切开的正确拓扑结构中。

2. 排除伪阴性（False Negatives Trap）：如果实验组（诱饵 + 真实靶蛋白NubG）没有任何信号，而 pOST1-NubI 阳性对照组也没有信号，说明整个 yeast 双杂交体系（或变性切开过程）出了故障，而非靶蛋白不结合，从而避免了科研误判。

3. 系统灵敏度校准：通过与 pOST1-NubG（突变体，作为阴性对照）并列平行实验，可清晰校准出当前测试酵母感受态菌株（如 NMY51）的背景噪音与筛选严谨度（Stringency）。

三 实验室质粒复苏、转化、扩增与保存标准步骤

1. 菌株与扩增培养基配置

• 大肠杆菌宿主：推荐使用常规克隆菌株，如 DH5a、TOP10 或 XL10-Gold。

• 大肠杆菌培养体系：LB 肉汤/固体琼脂培养基，添加最终工作浓度为 100 ug/mL 的氨苄青霉素（Ampicillin）。

• 酵母宿主与筛选体系：通常使用 NMY51 报告株，转化后的筛选体系为 SD-Leu 或者是 SD-Trp-Leu（双转）合成缺陷型培养基。

2. 质粒干粉/转化复苏步骤（Transformation & Recovery）

1. 若收到的是离心管内的质粒 DNA 干粉（或纸片），加入 20-50 uL 无菌去离子水或 TE 缓冲液，轻微震荡并静置 5 分钟，使其完全溶解。

2. 取出 50-100 uL 大肠杆菌 DH5a 感受态细胞置于冰上融化。

3. 加入 1-2 uL 溶解后的 pOST1-NubI 质粒 DNA，轻弹管底混匀，冰浴 30 分钟。

4. 将离心管置于 42 摄氏度水浴中精确热击 45-60 秒，随后立即置于冰上迅速冷却 2 分钟（切勿剧烈摇晃）。

5. 向管内加入 500 uL 不含抗生素的无菌 LB 肉汤（或 SOC 培养基），置于 37 摄氏度振荡孵育箱内，以 200 rpm 复苏匀速摇菌 45-60 分钟。

6. 以 4000 rpm 离心 3 分钟弃去部分上清，留约 100 uL 液体将菌体吹匀，涂布于含 100 ug/mL 氨苄青霉素的 LB 固体平板上。

7. 置于 37 摄氏度培养箱中倒置培养 14-16 小时，等待单菌落长出。

3. 质粒扩增与高纯度提取（Amplification & Extraction）

1. 从长有单菌落的氨苄平板上，挑选一个边缘清晰的健壮菌落，接种至 5-10 mL 含氨苄青霉素的 LB 液体肉汤试管中。

2. 置于 37 摄氏度、220 rpm 振荡培养 12 至 16 小时（过夜培养） 至对数生长晚期。

3. 利用标准的高纯度质粒提取试剂盒（Plasmid Miniprep Kit，基于硅胶柱离心法）收集菌体并提取质粒 DNA。用无菌无酸的灭菌水（或碱性 Elution Buffer）洗脱，利用分光光度计测量并记录浓度（A260/A280 比例应在 1.8-2.0 之间）。

4. 质粒及工程菌长期保存（Storage Options）

• 质粒 DNA 保存：提取出的纯化 pOST1-NubI 质粒可分装为小管，置于 -20 摄氏度 冰箱可稳定保存 1-2 年；若置于 -80 摄氏度 超低温冰箱中则可近乎无限期稳定保存。避免反复冻融。

• 大肠杆菌甘油菌保存：取扩增旺盛未完全衰亡的过夜菌液 800 uL，加入 200 uL 灭菌高纯无菌甘油（最终甘油浓度约 20%），在冻存管中彻底颠倒混匀，立即投入 -80 摄氏度 超低温冰箱内，可连续存活数年以上。

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.

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