BioVector® SPLICSFAST-LDh-MT-P2A 质粒 / SPLICSFAST-LDh-MT-P2A Plasmid (Addgene #214896)

一、 背景与来源说明 / Background & Origin

SPLICSFAST-LDh-MT-P2A（Addgene #214896）是一种基于 SPLICS（Split-GFP-based Contact Site Sensor）与 FAST 荧光标记技术设计的发光/荧光生物传感器质粒。该质粒专用于实时检测与量化细胞内脂滴（Lipid Droplets, LD）与线粒体（Mitochondria, MT）之间的膜接触位点（Organelle Contact Sites, MCS）。通过 P2A 自剪切肽序列，质粒可实现互补探针两端等摩尔量的共表达，用于线粒体-脂滴相互作用及脂质代谢调控的研究。

二、 质粒主要结构与生物学特征 / Biological Characteristics

• 质粒类型 / Vector Type：哺乳动物表达质粒 / 细胞器接触位点荧光传感器 (Mammalian Expression Vector / MCS Sensor)

• 宿主表达体系 / Expression Host：哺乳动物细胞 (Mammalian Cells)

• 细菌筛选标记 / Bacterial Selection：氨苄青霉素 (Ampicillin, 100 ug/mL)

• 核心元件构成 / Key Functional Components：

  • CMV / CAG 启动子：驱动高水平转录表达

  • LDh (Lipid Droplet targeting head)：靶向脂滴膜的定位信号

  • MT (Mitochondrial targeting sequence)：靶向线粒体外膜/基质的定位信号

  • SPLICSFAST 拆分荧光探针：当脂滴与线粒体相距在纳米级距离（如 8 至 50 nm）时发生复性并发出荧光信号

  • P2A 肽：核糖体跳跃/自剪切序列，保证 LD 和 MT 两个探针单质粒同步等比例表达

• 应用领域 / Applications：脂滴-线粒体接触位点 (LD-MT MCS) 动态成像、脂质代谢与脂肪重塑、自噬与线粒体质控研究

三、 培养条件与转化指南 / Culture & Handling Protocols

1. 细菌培养与筛选 / Bacterial Culture

• 培养基 / Broth & Agar：LB 液体/固体培养基

• 抗生素 / Antibiotics：100 ug/mL 氨苄青霉素 (Ampicillin)

• 克隆宿主 / E. coli Host：DH5alpha 或 TOP10 感受态细胞

2. 转化与细胞转染操作 / Transformation & Transfection

• 热休克转化 / Heat-shock Protocol：

  1. 取 DH5alpha 感受态细胞于冰上融化，加入 1 到 2 ug/uL 质粒 DNA 1 uL。

  2. 冰浴 30 分钟，42 摄氏度热休克 45 秒，立即置于冰上 2 分钟。

  3. 加入无抗 LB，37 摄氏度复苏 1 小时，涂布于 Amp 培养皿。

• 细胞转染 / Mammalian Transfection：使用 Lipofectamine 3000 或 PEI 转染哺乳动物细胞，转染后 24 至 48 小时可在荧光显微镜或高内涵成像仪下观测信号。

四、 技术指标简表 / Technical Data Summary

BioVector® SPLICSFAST-LDh-MT-P2A Plasmid (Addgene #214896)

1. Background and Origin

SPLICSFAST-LDh-MT-P2A (Addgene #214896) is a specialized fluorescent biosensor vector designed to detect and quantify membrane contact sites (MCS) between lipid droplets (LD) and mitochondria (MT) in living cells. Utilizing the SPLICSFAST split-reporter architecture combined with a P2A self-cleaving peptide, this single plasmid enables stoichiometric co-expression of organelle-targeted probe fragments to measure nanoscale organelle interactions.

2. Biological Characteristics

• Vector Type: Mammalian Expression Vector / Organelle Contact Site Sensor

• Expression Host: Mammalian cells

• Bacterial Selection: Ampicillin (100 ug/mL)

• Key Functional Components:

  • CMV / CAG Promoter: Drives robust expression in eukaryotic hosts

  • LDh: Lipid droplet-targeting motif

  • MT: Mitochondrial outer membrane or matrix-targeting signal

  • SPLICSFAST Split Probe: Reconstitutes fluorescence when LD and MT are within nanoscale proximity (approx. 8 to 50 nm)

  • P2A Peptide: Facilitates ribosomal skipping to produce equal molar ratios of both organelle-targeted moieties from a single transcript

• Applications: Live-cell organelle contact site (LD-MT MCS) quantification, lipid trafficking, metabolic flux studies, and organelle crosstalk analysis

3. Culture and Handling Protocols

Bacterial Culture and Selection

• Growth Medium: LB Broth / LB Agar

• Selective Antibiotic: Ampicillin at 100 ug/mL

• Cloning Host: E. coli DH5alpha or TOP10

Transformation and Cell Transfection

• Transformation Protocol:

  1. Thaw DH5alpha competent cells on ice and add 1 to 2 uL of plasmid DNA.

  2. Incubate on ice for 30 minutes, heat-shock at 42 degrees Celsius for 45 seconds, then return to ice for 2 minutes.

  3. Outgrow in antibiotic-free LB broth for 1 hour at 37 degrees Celsius, then spread onto selective Ampicillin plates.

• Transfection: Transfect mammalian cells using standard reagents (e.g., Lipofectamine 3000 or PEI). Perform fluorescent imaging 24 to 48 hours post-transfection.

4. Technical Data Summary

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