K12 Hfr BioVector® 大肠杆菌菌株 BioVector® E.coli K12 Hfr Strain

BioVector® 大肠杆菌 K12 Hfr 菌株

BioVector® E.coli K12 Hfr Strain Text-Based Datasheet

1 产品基本信息与遗传背景 / Product Identification and Genetic Background

• 产品名称 (Product Name)：BioVector® 大肠杆菌 K12 Hfr 基因高频重组工程菌株 (BioVector® E.coli K12 Hfr High Frequency of Recombination Strain)

• 常用别名 (Synonyms)：BioVector® K12 Hfr，BioVector® Hfr Cavalli，BioVector® Hfr Hayes

• 生物学分类 (Organism Source)：大肠杆菌 Escherichia coli（K12 衍生谱系）

• 基本生长特性 (Growth Properties)：原核生物悬浮与固体板生长。在液体培养基中呈均匀混浊悬浮生长；在固体琼脂平板上形成圆形、边缘整齐、表面光滑的典型大肠杆菌菌落。(Prokaryotic cell suspension or solid agar plating. Grows as a homogeneous turbid suspension in liquid media, and forms circular, smooth, entire-edged colonies on standard agar plates.)

• Hfr 构造特征机制 (Hfr Mechanism)：该菌株属于基因高频重组株（High Frequency of Recombination）。通过自发或人工定向选择，致育因子（F质粒 F plasmid）已经完全整合到了大肠杆菌 K12 的染色体基因组中。当与 F减（F-minus，不含 F 质粒的受体菌）进行接合生殖（Conjugation）时，整合的 F 因子会驱动整个宿主染色体以极高的频率线性转移至受体菌中，启动基因重组。(Classified as a High Frequency of Recombination strain. Through targeted selection, the fertility factor or F plasmid has integrated entirely into the main E.coli K12 chromosomal genome. During bacterial conjugation with an F-minus recipient, the integrated F factor drives the directional, linear transfer of the host chromosome into the recipient at an exceptionally high rate, initiating genetic recombination.)

• 核心科研价值 (Core Research Significance)：BioVector® 大肠杆菌 K12 Hfr 是经典遗传学、细菌基因组绘图、以及现代生物工程中不可或缺的标杆菌株。利用该菌株与不同标记的受体菌进行“中断接合实验（Interrupted mating experiment）”，科研人员能够根据基因进入受体菌的时间先后，精准绘制大肠杆菌染色体的相对物理位点（以分钟为单位的基因图谱）。它也是研究原核生物水平基因转移、重组修复系统（recA、recBCD 通路）生理机制的核心底盘工具。(BioVector® E.coli K12 Hfr stands as an indispensable benchmark strain in classical genetics, bacterial genomic mapping, and modern bioengineering. By performing interrupted mating experiments with marked F-minus strains, researchers map the relative physical coordinates of the E.coli chromosome in minutes based on gene entry times. It is also a premier tool for exploring horizontal gene transfer and prokaryotic recombination repair pathways.)

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2 细胞生物学特征与核心重组表型 / Cellular Properties and Recombination Profiles

BioVector® 大肠杆菌 K12 Hfr 在维持期表现为标准的原核细胞分裂，在接合期展现出定向的染色体供体特征：The BioVector® E.coli K12 Hfr line functions as a standard dividing prokaryote during maintenance, but acts as a directional chromosomal donor during conjugation:

营养生长状态 (Vegetative Proliferation State)

在无压力或无受体菌存在时，菌株执行标准的二分裂方式（Binary fission）快速扩增。在富营养培养基中，倍增时间通常在 20 到 30 分钟之间。In the absence of recipient strains, the culture undergoes standard binary fission for rapid biomass expansion, with doubling times ranging between 20 and 30 minutes in rich media.

接合与基因转移特征 (Conjugation and Gene Transfer Dynamics)

当与受体菌混合后，细胞表面迅速形成特异性的性菌毛（Sex pili），启动单向的 DNA 复制与转移。其核心特征包括：Upon blending with an F-minus population, the strain constructs surface sex pili to initiate unidirectional DNA replication and rolling-circle transfer. Its attributes include:

• 转移起始位点固定 (Fixed Origin of Transfer)：DNA 转移从 F 因子整合位点内部的特定起点（oriT）开始，以预先固定的方向（顺时针或逆时针，依具体 Hfr 亚型如 HfrH 或 HfrC 而定）线性输入受体菌。(DNA transfer initiates at a specific origin of transfer inside the integrated F sequence, rolling linearly into the recipient in a fixed orientation depending on the sub-strain genotype.)

• 高频重组率 (High Recombination Rate)：其诱导受体菌发生染色体基因重组的频率比普通 F加（F-plus）游离质粒菌株高出数百倍到上千倍，但由于接合过程中管道极易断裂，受体菌极少能获得完整的 F 因子序列，因此接合后的受体菌绝大多数仍保持 F减 表型。(The recombination frequency is hundreds of times higher than standard F-plus strains. However, due to spontaneous pili disruption during mating, recipients rarely secure the terminal F factor sequence, meaning post-conjugation recipients predominantly remain F-minus.)

• 菌株特异性遗传标记 (Genetic Markers)：通常带有明确的营养缺陷型或抗生素耐药性标记（如带有特定的大肠杆菌糖代谢、氨基酸合成缺陷），便于接合实验后的选择性平板筛选。(Typically carries well-defined auxotrophic or antibiotic resistance markings to streamline selective plating readouts after mating sequences.)

3 专用培养基配方规范 / Culturing Medium Formulations

警告 / Critical WarningBioVector® 大肠杆菌 K12 Hfr 菌株在进行接合实验前，必须使用未添加任何选择性抗生素的富营养培养基使其处于对数生长期。如果在维持阶段错误添加了非目标耐药性抗生素，极易诱发 F 因子的自发剪切脱落，使其退化为普通菌株。BioVector® E.coli K12 Hfr must be grown into log-phase using rich media free of non-target selective antibiotics prior to mating assays. Incorrect exposure to counter-selective antibiotics can trigger spontaneous excision of the F factor, reverting the line into a standard phenotype.

A 阶段：菌株日常扩增与维持培养基（用于高活力生物量扩增）

Phase A: Proliferating Maintenance Medium (For High-Viability Biomass Expansion)

• 基础液体培养基 (Liquid Medium)：BioVector® LB Liquid Medium 液体培养基（每升含 10 g 胰蛋白胨、5 g 酵母提取物、10 g 氯化钠，无菌高压灭菌 / containing 10 grams Tryptone, 5 grams Yeast Extract, and 10 grams NaCl per liter）。

• 基础固体培养基 (Solid Agar Plate)：BioVector® LB Agar Medium 固体琼脂培养基（在上述成分基础上添加 1.5% 琼脂粉）。

• 选择性添加剂 (Selective Additives)：根据特定亚型克隆的特殊基因型，添加适量 BioVector® 链霉素或特定氨基酸补剂以维持遗传纯度。(Supplement with targeted BioVector® Streptomycin or specified amino acids depending on the exact sub-clonal genotype to maintain strain purity.)

B 阶段：基因接合与中断实验培养基（用于选择性重组子筛分）

Phase B: Interrupted Mating and Selection Medium (For Recombinant Colony Isolation)

• 固体筛选骨架 (Selective Matrix)：BioVector® M9 Minimal Salts Agar 最小盐固体基础培养基（不含任何碳源与氨基酸）。

• 定制碳源与筛选微量组件 (Custom Carbons and Micro-Supplements)：

  • BioVector® Glucose (葡萄糖溶液，20%)：过滤除菌，作为基础碳源。

  • BioVector® Specific Amino Acids (特异性氨基酸组合补剂)：根据供体 Hfr 菌与受体 F减 菌的营养缺陷型差异，缺失添加特定氨基酸，用以选择性杀灭供体菌并仅允许重组成功的受体菌长出菌落。(Omit targeted amino acids based on cross-auxotrophic gaps between donor and recipient strains to selectively counter-select parent stocks and resolve functional recombinants.)

4 物理环境与接合反应控制参数 / Environmental Controls and Mating Parameters

• 培养与扩增温度 (Growth Temperature)：37.0 摄氏度（Constant temperature at 37.0 degrees Celsius for optimal replication kinetics）。

• 氧气流体力学控制 (Oxygen Fluid Dynamics)：

  • 日常扩增维持：需在恒温调速摇床中以 每分钟 200 到 250 转（200 到 250 rpm） 的速度进行强力振荡培养，以保证充足的溶氧量。(For routine expansion, shake vigorously at 200 to 250 rpm to maintain high dissolved oxygen pathways.)

  • 接合反应阶段 (Conjugation Phase - Critical)：当供体 Hfr 菌与受体 F减 菌按比例混合后，必须立即停止摇床摇动，将混合液置于 37 摄氏度孵箱中进行绝对静置培养（0 rpm）。任何轻微的液体摇晃或机械剪切力都会瞬间折断纤细的性菌毛，导致染色体转移过程提前中断。 (During donor-recipient mating, the mixture must be incubated under absolute static conditions at 0 rpm. Even minor fluid turbulence or mechanical shear will break the delicate sex pili, prematurely interrupting the chromosome transfer cascade.)

5 菌株传代、保存与接合功能操作规范 / Subculturing and Conjugation Protocols

菌株日常传代与活化 / Routine Passaging and Strain Activation

• 平板划线维持：每隔 2 到 3 周，应从 4 摄氏度暂存板上挑取单菌落重新划线至新鲜的 BioVector® LB 琼脂平板上。

• 液体接种稀释比例：按 1比100 的体积比将活化后的单菌落接种至液体 LB 中，37 摄氏度振荡培养过夜（约 12 到 16 小时）即可达到饱和期。

经典中断接合实验操作流程 / Classic Interrupted Mating Protocol

1. 分别将 BioVector® 大肠杆菌 K12 Hfr 供体菌（例如大肠杆菌链霉素敏感、苏氨酸和亮氨酸不缺陷株）与合适的 F减 受体菌（例如大肠杆菌链霉素耐药、苏氨酸和亮氨酸缺陷株），接种于 BioVector® 液体 LB 培养基中，37 摄氏度摇床培养至对数生长中期（每毫升约有 2.0乘以10的8次方 个活菌）。(Grow both the BioVector® Hfr donor and F-minus recipient into mid-log phase using clear LB broth.)

2. 取 1.0 mL 供体菌液与 9.0 mL 受体菌液混合于无菌大试管中（混合比例通常为 1比9，确保受体菌过量），轻轻混匀。(Mix 1.0 mL donor with 9.0 mL recipient in a sterile tube at a 1:9 ratio to ensure excessive recipient counts.)

3. 立即将试管置于 37 摄氏度水浴或孵箱中静置培养（0 rpm），正式开始接合记时。(Place the mixture statically inside a 37 degrees Celsius water bath; start timing the conjugation sequence.)

4. 根据基因图谱测定需求，在接合开始后的第 5 分钟、10 分钟、15 分钟、20 分钟等设定时间点，分别吸取 0.5 mL 混合液，立刻投入冰预冷的无菌小管中，并使用涡旋振荡器（Vortex）在最高速度下剧烈猛烈振荡 60 秒。利用剧烈的机械剪切力强行成片折断性菌毛，终止基因继续转移。(At set temporal intervals, draw 0.5 mL samples, transfer into ice-cold vials, and vortex violently at maximum speed for 60 seconds to shear sex pili and stop further transfer.)

5. 将剧烈振荡后的样品进行适当梯度稀释，全量涂布于含有链霉素且缺失苏氨酸/亮氨酸的 BioVector® M9 最小盐选择性琼脂平板 上。(Perform serial dilutions and plate onto BioVector® M9 selective agar containing streptomycin but lacking leucine/threonine.)

6. 将平板倒置于 37 摄氏度孵箱培养 24 到 48 小时。计算各个时间点长出的重组子菌落数量，即可根据菌落首次出现的时间，换算出目标基因在染色体基因组上的相对物理分钟位置。(Incubate plates at 37 degrees Celsius for 24 to 48 hours. Map the colony counts against transfer times to chart the relative genomic location of the target loci in minutes.)

6 甘油菌冻存复苏与长期保存 / Cryopreservation and Revitalization Workflow

1. 甘油冷冻管配制：在超净台内，吸取 800微升 处于对数生长旺盛期的 BioVector® Hfr 液体菌液，加入到盛有 200微升 无菌无毒纯甘油的冷冻管中（最终甘油体积分数达到 20%），彻底颠倒摇匀。

2. 配制完成后，将冷冻管直接投入零下 80 摄氏度超低温冰箱中，可稳定保存 2 到 3 年；长期永久保存建议放置于液氮气相环境中。(Store engineered glycerol stocks directly inside a minus 80 degrees Celsius freezer for stable banking spanning 2 to 3 years; move to liquid nitrogen vapor grids for permanent repository archiving.)

3. 冻存回收活化：从零下 80 摄氏度冰箱中取出冻存管，置于冰盒上微融。在超净台内使用无菌接种环或枪头，直接刮取冰霜表层少许菌体，在预热的 BioVector® LB 固体平板 上进行功能划线，随后将冻存管迅速放回深冷冰箱，严禁发生反复彻底冻融。(Retrieve the vial and place on ice. Use a sterile loop to scrape the frozen surface crust, streak directly onto a BioVector® LB plate, and return the vial to the deep freezer immediately to prevent cycle-thawing.)

4. 将划线平板置于 37 摄氏度孵箱培养 16 到 24 小时，即可获得高活力的纯系单菌落。

7 生物安全、纯度控制与质控规范 / Biosafety, Purity Controls and Quality Assays

• 生物安全级别 (Biosafety Level)：BSL-1。大肠杆菌 K12 谱系属于国际公认的非致病性安全实验室常规工程菌，无人类感染和扩散风险。日常操作执行基础无菌规范，实验残液及平板在废弃前需执行 121 摄氏度高压蒸汽灭菌 30 分钟。(BSL-1 classification. The E.coli K12 lineage represents a globally validated non-pathogenic laboratory vector framework; route spent broths and plates through standard institutional autoclaving lines at 121 degrees Celsius for 30 minutes before waste consolidation.)

• F 因子整合状态定期抽检（质控红线）/ Functional Recombination Quality Check：整合在宿主染色体上的 F 因子存在极低的自发自解离概率。如果自发解离并带有宿主部分基因，则退化为 F一撇（F-prime）表型；如果完全解离则退化为普通 F加。若连续传代或保存不当，Hfr 菌株将彻底丧失高频定向转移整段染色体的表型。 严格质控表现为：每隔 6 个月或每批量配制甘油库时，必须抽样一管与标准的已知缺陷型 F减 受体菌进行交配对照测试。如果测试发现其在 15 分钟内无法驱动前位标记基因发生高频重组转导（重组效率低于初始质控基准的一个数量级），则证实该批菌株已经发生严重降解退化，必须全盘销毁，并重新向原厂申请低传代原始原始菌种进行恢复。 (The chromosomally integrated F factor undergoes spontaneous loop-out excision at low rates. Over extended cycles, Hfr stocks can slide into F-prime or F-plus variants, destroying the unique capacity for directional chromosome transfer. To enforce strict quality control, sample the seed stock every 6 months via a control mating assay with a known F-minus recipient. If the strain fails to deliver the expected gene transfer efficiency within 15 minutes, the lot has drifted; destroy active slants immediately and restore from a verified master stock vial.)

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