BioVector® GS1783, a specialised Escherichia coli strain engineered by Gregory Smith for advanced BAC (Bacterial Artificial Chromosome) recombineering using the Red/ET system.

BioVector® GS1783 大肠杆菌重组工程菌株技术档案 / BioVector® GS1783 E. coli Recombineering Strain Datasheet

一、 背景与来源说明

GS1783 是专用于无痕重组工程（Scarless En Passant Recombineering）的大肠杆菌（E. coli）工程菌株，由 Nikolaus Osterrieder 实验室基于 E. coli DH10B 改造建立。该菌株染色体整合了受温敏型阻抑蛋白（$cI857$）控制的 $\lambda$ Red 重组系统（$\gamma, \beta, exo$），以及受 L-阿拉伯糖诱导启动子（$P_{BAD}$）控制的 I-SceI 归巢内切酶（Homing Endonuclease）。GS1783 是对细菌人工染色体（BAC）、P1 衍生人工染色体（PAC）及大型病毒基因组（如疱疹病毒、痘病毒）进行高效率无痕点突变、基因敲除、片段插入及精准修饰的金标准宿主菌。

二、 基因型与核心机制

1. 菌株染色体基因型

DH10B derivative: F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) npr+ [cI857 Δ(cro-bioA)::λ Red-PBAD-I-SceI]

2. En Passant 无痕重组工作原理

• 第一步：Red 介导的插入（Primary Recombination）

  • 在 42°C 下短暂热激诱导表达 $\lambda$ Red 重组酶，将含有抗性标记（如 $Kan^R$）、侧翼 I-SceI 识别位点及两侧带有相同同源重复序列（Internal Duplication / Internal Homology）的 PCR 产物重组整合至 BAC 靶位点。

• 第二步：I-SceI 切割与无痕切除（Secondary Recombination）

  • 添加 L-阿拉伯糖（L-Arabinose）诱导表达 I-SceI 内切酶，特异性引入双链断裂（DSB）。结合第二次 42°C 热激，促使菌株利用靶位点的内部同源重复序列发生分子内同源重组，精准切除抗性选择标记，完全不遗留任何异源序列（如 LoxP 或 FRT 疤痕）。

三、 培养条件与无痕重组诱导指南

1. 基础培养与保存条件

• 常规培养温度：30°C（日常维持与扩增切勿超过 34°C，防止 $cI857$ 阻抑蛋白失活导致 Red 重组酶提前泄漏而引发质粒/基因组重组不稳定）。

• 基础抗性：链霉素（Streptomycin, 50 $\mu\text{g/mL}$，源自染色体 $rpsL$ 突变）。

• 保存条件：含 20% 甘油的 LB 菌液于 -80°C 深低温保存。

2. 标准无痕重组操作流程

1. Red 重组与电转化：

   • 将含有目标 BAC 的 GS1783 菌株于 30°C 培养至 $OD_{600} \approx 0.5 - 0.7$。

   • 置于 42°C 水浴振荡热激 15 分钟 以诱导表达 Red 重组酶。

   • 冰浴冰镇 10–15 分钟后，用冰预冷的 10% 甘油洗涤 3 次，制备电转化感受态细胞并电转 PCR 重组片段。

2. I-SceI 切割与标记切除：

   • 挑选第一步成功重组的阳性克隆，接种于含 1% L-阿拉伯糖（L-Arabinose） 的无糖 LB 液体培养基中，30°C 振荡培养 1 小时（诱导 I-SceI 切割）。

   • 再次转入 42°C 水浴热激 15 分钟（启动第二次分子内重组）。

   • 30°C 继续复苏培养 1–2 小时，稀释涂布于无抗性 LB 平板上。

   • 通过平板影印（Replica Plating）筛选抗性丧失（如 $Kan^S$）的无痕突变阳性克隆。

四、 技术指标简表

BioVector® GS1783 E. coli Recombineering Strain Datasheet

1. Background & Context

The E. coli strain GS1783 is a specialized host designed for scarless En Passant recombineering in Bacterial Artificial Chromosomes (BACs), P1-derived Artificial Chromosomes (PACs), and large viral genomes (e.g., herpesvirus, poxvirus). Developed in Nikolaus Osterrieder's laboratory from E. coli DH10B, GS1783 features chromosome-integrated $\lambda$ Red recombination genes ($\gamma, \beta, exo$) controlled by the temperature-sensitive $cI857$ repressor, alongside an L-arabinose-inducible I-SceI homing endonuclease gene under the $P_{BAD}$ promoter. It represents the industry standard for performing precise point mutations, insertions, deletions, and tag integrations without leaving residual scar sequences.

2. Genotype & Recombineering Mechanism

2.1 Chromosomal Genotype

DH10B derivative: F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) npr+ [cI857 Δ(cro-bioA)::λ Red-PBAD-I-SceI]

2.2 Principles of En Passant Mutagenesis

• Step 1: Primary Red-Mediated Insertion

  • A brief heat shock at 42°C induces $\lambda$ Red recombinase to integrate a PCR fragment containing a selection marker (e.g., $Kan^R$), an adjacent I-SceI cleavage site, and flanking internal duplications (internal homology) into the target BAC locus.

• Step 2: I-SceI Cleavage & Secondary Excision

  • Addition of L-arabinose induces I-SceI endonuclease expression, introducing a site-specific double-strand break (DSB). A second 42°C heat shock then activates Red-mediated intramolecular recombination between the internal repeats, seamlessly excising the selection cassette without leaving scar sequences (such as LoxP or FRT).

3. Culture & Induction Protocols

3.1 Growth Conditions & Antibiotic Maintenance

• Routine Incubation Temperature: 30°C (Do NOT incubate above 34°C during routine culture to prevent premature activation of the $\lambda$ Red recombinase).

• Selective Antibiotic: Streptomycin (50 $\mu\text{g/mL}$, encoded by chromosomal $rpsL$).

• Storage: Store in LB containing 20% glycerol at -80°C.

3.2 En Passant Recombineering Workflow

1. Primary Recombination & Electroporation:

   • Culture GS1783 harboring the target BAC at 30°C to $OD_{600} \approx 0.5 - 0.7$.

   • Transfer to a 42°C water bath for 15 minutes with shaking to induce Red recombinase.

   • Chill on ice for 10–15 min, wash 3 times with ice-cold 10% glycerol, and electroporate with the purified PCR product.

2. I-SceI Induction & Marker Resolution:

   • Inoculate positive primary recombinants into glucose-free LB medium containing 1% L-Arabinose and shake at 30°C for 1 hour.

   • Transfer to a 42°C water bath for 15 minutes to induce secondary intramolecular recombination.

   • Outgrow at 30°C for 1–2 hours, then spread diluted cultures onto non-selective LB agar plates. Screen for marker loss (e.g., $Kan^S$) via replica plating.

4. Technical Data Summary

• • $\lambda$