BioVector® SW102 大肠杆菌重组工程菌株技术档案 / BioVector® SW102 E. coli Recombineering Strain Datasheet

一、 背景与来源说明

SW102 是一种专用于细菌人工染色体（BAC）及大型 DNA 片段重组工程（Recombineering）的大肠杆菌（E. coli）工程菌株，由 National Cancer Institute (NCI) 的 Neal Copeland 和 Nancy Jenkins 实验室基于 E. coli DH10B 改造构建。该菌株在染色体上整合了受温敏型阻抑蛋白（$cI857$）严格调控的 $\lambda$ Red 重组系统（$\gamma, \beta, exo$），并敲除了 $galK$ 基因（半乳糖激酶）。SW102 是利用 $galK$ 正/负向选择标记系统（GalK Positive/Negative Selection System） 对 BAC 进行高效率点突变、无痕修饰及无限制性内切酶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: [mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) npr+] ΔgalK [cI857 Δ(cro-bioA)::λ Red]

2. galK 双向筛选重组工作原理

• 第一步：正向选择（Positive Selection，引入 $galK$ 盒）

  • 将包含 $galK$ 表达盒及目标位点上下游同源臂（~50 bp）的 PCR 产物电转入经 42°C 热激诱导表达 Red 重组酶的 SW102 菌株中。

  • 涂布于以半乳糖为唯一碳源的最小培养基（M63 / Galactose Minimal Medium）上，仅重组整合了 $galK$ 的阳性克隆能够生长。

• 第二步：负向选择（Negative Selection，无痕替换/切除 $galK$）

  • 将包含目标点突变或片段插入的寡核苷酸单链（ssDNA）/双链（dsDNA）电转入二次热激的 SW102 菌株中。

  • 涂布于含有 2-脱氧-D-半乳糖（2-Deoxy-D-galactose, 2-DOG） 和甘油的最小培养基上。GalK 酶会将 2-DOG 磷酸化生成有毒的 2-deoxy-galactose-1-phosphate 杀死未重组菌株，仅 $galK$ 被精准替换/切除的无痕阳性克隆 能够存活。

三、 培养条件与重组操作指南

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. 标准重组与感受态制备

1. Red 重组酶热激诱导：

   • 将 SW102 接种于 LB（或 MacConkey 基础培养基）中，30°C 振荡培养至 $OD_{600} \approx 0.4 - 0.6$。

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

2. 电转化感受态制备：

   • 冰浴冰镇 10–15 分钟后，用冰预冷的无菌水或 10% 甘油离心洗涤 3 次，快速制备电转化感受态细胞。

3. 电转化与筛选：

   • 加入 DNA 片段（50–100 ng 双链片段或 100–250 ng 单链寡核苷酸），电击后加入 SOC/LB 于 30°C 复苏 1–1.5 小时，涂布于对应选择性培养基（M63/Gal 最小培养基或含 2-DOG 培养基）进行正/负向筛选。

四、 技术指标简表

BioVector® SW102 E. coli Recombineering Strain Datasheet

1. Background & Context

The E. coli strain SW102 is a specialized host engineered for Bacterial Artificial Chromosome (BAC) recombineering and large genomic fragment modifications. Developed by Neal Copeland and Nancy Jenkins at the National Cancer Institute (NCI) from E. coli DH10B, SW102 harbors a temperature-sensitive $\lambda$ Red recombination system ($\gamma, \beta, exo$) integrated into its chromosome under the control of the $cI857$ repressor, along with a specific deletion of the $galK$ gene (galactokinase). SW102 represents the cornerstone strain for executing seamless point mutations, small deletions, and tag insertions in BACs using the GalK Positive/Negative Selection System.

2. Genotype & Recombineering Mechanism

2.1 Chromosomal Genotype

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

2.2 Principles of galK Counter-Selection Recombineering

• Step 1: Positive Selection (Insertion of $galK$ Cassette)

  • A PCR cassette containing the $galK$ gene flanked by ~50 bp target homology arms is electroporated into 42°C heat-induced SW102.

  • Cells are plated on M63 minimal medium containing galactose as the sole carbon source. Only recombinants expressing GalK survive.

• Step 2: Negative Selection (Seamless Replacement/Removal of $galK$)

  • A synthetic single-stranded oligonucleotide (ssDNA) or double-stranded DNA (dsDNA) containing the desired point mutation/modification is electroporated into re-induced SW102 cells.

  • Cells are plated on minimal medium containing 2-deoxy-D-galactose (2-DOG) and glycerol. GalK phosphorylates 2-DOG into toxic 2-deoxy-galactose-1-phosphate, killing un-recombined parent cells. Only seamless recombinants where $galK$ was completely replaced survive.

3. Culture & Handling 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 Recombineering & Electrocompetent Cell Preparation

1. $\lambda$ Red Recombinase Induction:

   • Inoculate SW102 containing the target BAC in LB or MacConkey base medium and shake at 30°C until $OD_{600} \approx 0.4 - 0.6$.

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

2. Electrocompetent Cell Preparation:

   • Chill on ice for 10–15 min, wash 3 times with ice-cold sterile water or 10% glycerol, and concentrate for electroporation.

3. Electroporation & Selection:

   • Pulse with 50–100 ng dsDNA or 100–250 ng ssDNA targeting oligo. Recover in SOC/LB at 30°C for 1–1.5 hours before plating on M63 Galactose minimal agar (Positive selection) or 2-DOG agar (Negative selection).

4. Technical Data Summary

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