BioVector? 大腸桿菌 K12 Hfr 菌株

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

1 產(chǎn)品基本信息與遺傳背景 / Product Identification and Genetic Background

• 產(chǎn)品名稱 (Product Name)：BioVector? 大腸桿菌 K12 Hfr 基因高頻重組工程菌株 (BioVector? E.coli K12 Hfr High Frequency of Recombination Strain)

• 常用別名 (Synonyms)：BioVector? K12 Hfr，BioVector? Hfr Cavalli，BioVector? Hfr Hayes

• 生物學(xué)分類 (Organism Source)：大腸桿菌 Escherichia coli（K12 衍生譜系）

• 基本生長特性 (Growth Properties)：原核生物懸浮與固體板生長。在液體培養(yǎng)基中呈均勻混濁懸浮生長；在固體瓊脂平板上形成圓形、邊緣整齊、表面光滑的典型大腸桿菌菌落。(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 構(gòu)造特征機(jī)制 (Hfr Mechanism)：該菌株屬于基因高頻重組株（High Frequency of Recombination）。通過自發(fā)或人工定向選擇，致育因子（F質(zhì)粒 F plasmid）已經(jīng)完全整合到了大腸桿菌 K12 的染色體基因組中。當(dāng)與 F減（F-minus，不含 F 質(zhì)粒的受體菌）進(jìn)行接合生殖（Conjugation）時，整合的 F 因子會驅(qū)動整個宿主染色體以極高的頻率線性轉(zhuǎn)移至受體菌中，啟動基因重組。(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.)

• 核心科研價值 (Core Research Significance)：BioVector? 大腸桿菌 K12 Hfr 是經(jīng)典遺傳學(xué)、細(xì)菌基因組繪圖、以及現(xiàn)代生物工程中不可或缺的標(biāo)桿菌株。利用該菌株與不同標(biāo)記的受體菌進(jìn)行“中斷接合實驗（Interrupted mating experiment）”，科研人員能夠根據(jù)基因進(jìn)入受體菌的時間先后，精準(zhǔn)繪制大腸桿菌染色體的相對物理位點（以分鐘為單位的基因圖譜）。它也是研究原核生物水平基因轉(zhuǎn)移、重組修復(fù)系統(tǒng)（recA、recBCD 通路）生理機(jī)制的核心底盤工具。(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 細(xì)胞生物學(xué)特征與核心重組表型 / Cellular Properties and Recombination Profiles

BioVector? 大腸桿菌 K12 Hfr 在維持期表現(xiàn)為標(biāo)準(zhǔn)的原核細(xì)胞分裂，在接合期展現(xiàn)出定向的染色體供體特征：The BioVector? E.coli K12 Hfr line functions as a standard dividing prokaryote during maintenance, but acts as a directional chromosomal donor during conjugation:

營養(yǎng)生長狀態(tài) (Vegetative Proliferation State)

在無壓力或無受體菌存在時，菌株執(zhí)行標(biāo)準(zhǔn)的二分裂方式（Binary fission）快速擴(kuò)增。在富營養(yǎng)培養(yǎng)基中，倍增時間通常在 20 到 30 分鐘之間。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.

接合與基因轉(zhuǎn)移特征 (Conjugation and Gene Transfer Dynamics)

當(dāng)與受體菌混合后，細(xì)胞表面迅速形成特異性的性菌毛（Sex pili），啟動單向的 DNA 復(fù)制與轉(zhuǎn)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:

• 轉(zhuǎn)移起始位點固定 (Fixed Origin of Transfer)：DNA 轉(zhuǎn)移從 F 因子整合位點內(nèi)部的特定起點（oriT）開始，以預(yù)先固定的方向（順時針或逆時針，依具體 Hfr 亞型如 HfrH 或 HfrC 而定）線性輸入受體菌。(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.)

• 高頻重組率 (High Recombination Rate)：其誘導(dǎo)受體菌發(fā)生染色體基因重組的頻率比普通 F加（F-plus）游離質(zhì)粒菌株高出數(shù)百倍到上千倍，但由于接合過程中管道極易斷裂，受體菌極少能獲得完整的 F 因子序列，因此接合后的受體菌絕大多數(shù)仍保持 F減 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.)

• 菌株特異性遺傳標(biāo)記 (Genetic Markers)：通常帶有明確的營養(yǎng)缺陷型或抗生素耐藥性標(biāo)記（如帶有特定的大腸桿菌糖代謝、氨基酸合成缺陷），便于接合實驗后的選擇性平板篩選。(Typically carries well-defined auxotrophic or antibiotic resistance markings to streamline selective plating readouts after mating sequences.)

3 專用培養(yǎng)基配方規(guī)范 / Culturing Medium Formulations

警告 / Critical WarningBioVector? 大腸桿菌 K12 Hfr 菌株在進(jìn)行接合實驗前，必須使用未添加任何選擇性抗生素的富營養(yǎng)培養(yǎng)基使其處于對數(shù)生長期。如果在維持階段錯誤添加了非目標(biāo)耐藥性抗生素，極易誘發(fā) F 因子的自發(fā)剪切脫落，使其退化為普通菌株。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 階段：菌株日常擴(kuò)增與維持培養(yǎng)基（用于高活力生物量擴(kuò)增）

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

• 基礎(chǔ)液體培養(yǎng)基 (Liquid Medium)：BioVector? LB Liquid Medium 液體培養(yǎng)基（每升含 10 g 胰蛋白胨、5 g 酵母提取物、10 g 氯化鈉，無菌高壓滅菌 / containing 10 grams Tryptone, 5 grams Yeast Extract, and 10 grams NaCl per liter）。

• 基礎(chǔ)固體培養(yǎng)基 (Solid Agar Plate)：BioVector? LB Agar Medium 固體瓊脂培養(yǎng)基（在上述成分基礎(chǔ)上添加 1.5% 瓊脂粉）。

• 選擇性添加劑 (Selective Additives)：根據(jù)特定亞型克隆的特殊基因型，添加適量 BioVector? 鏈霉素或特定氨基酸補(bǔ)劑以維持遺傳純度。(Supplement with targeted BioVector? Streptomycin or specified amino acids depending on the exact sub-clonal genotype to maintain strain purity.)

B 階段：基因接合與中斷實驗培養(yǎng)基（用于選擇性重組子篩分）

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

• 固體篩選骨架 (Selective Matrix)：BioVector? M9 Minimal Salts Agar 最小鹽固體基礎(chǔ)培養(yǎng)基（不含任何碳源與氨基酸）。

• 定制碳源與篩選微量組件 (Custom Carbons and Micro-Supplements)：

  • BioVector? Glucose (葡萄糖溶液，20%)：過濾除菌，作為基礎(chǔ)碳源。

  • BioVector? Specific Amino Acids (特異性氨基酸組合補(bǔ)劑)：根據(jù)供體 Hfr 菌與受體 F減 菌的營養(yǎng)缺陷型差異，缺失添加特定氨基酸，用以選擇性殺滅供體菌并僅允許重組成功的受體菌長出菌落。(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 物理環(huán)境與接合反應(yīng)控制參數(shù) / Environmental Controls and Mating Parameters

• 培養(yǎng)與擴(kuò)增溫度 (Growth Temperature)：37.0 攝氏度（Constant temperature at 37.0 degrees Celsius for optimal replication kinetics）。

• 氧氣流體力學(xué)控制 (Oxygen Fluid Dynamics)：

  • 日常擴(kuò)增維持：需在恒溫調(diào)速搖床中以 每分鐘 200 到 250 轉(zhuǎn)（200 到 250 rpm） 的速度進(jìn)行強(qiáng)力振蕩培養(yǎng)，以保證充足的溶氧量。(For routine expansion, shake vigorously at 200 to 250 rpm to maintain high dissolved oxygen pathways.)

  • 接合反應(yīng)階段 (Conjugation Phase - Critical)：當(dāng)供體 Hfr 菌與受體 F減 菌按比例混合后，必須立即停止搖床搖動，將混合液置于 37 攝氏度孵箱中進(jìn)行絕對靜置培養(yǎng)（0 rpm）。任何輕微的液體搖晃或機(jī)械剪切力都會瞬間折斷纖細(xì)的性菌毛，導(dǎo)致染色體轉(zhuǎn)移過程提前中斷。 (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 菌株傳代、保存與接合功能操作規(guī)范 / Subculturing and Conjugation Protocols

菌株日常傳代與活化 / Routine Passaging and Strain Activation

• 平板劃線維持：每隔 2 到 3 周，應(yīng)從 4 攝氏度暫存板上挑取單菌落重新劃線至新鮮的 BioVector? LB 瓊脂平板上。

• 液體接種稀釋比例：按 1比100 的體積比將活化后的單菌落接種至液體 LB 中，37 攝氏度振蕩培養(yǎng)過夜（約 12 到 16 小時）即可達(dá)到飽和期。

經(jīng)典中斷接合實驗操作流程 / Classic Interrupted Mating Protocol

1. 分別將 BioVector? 大腸桿菌 K12 Hfr 供體菌（例如大腸桿菌鏈霉素敏感、蘇氨酸和亮氨酸不缺陷株）與合適的 F減 受體菌（例如大腸桿菌鏈霉素耐藥、蘇氨酸和亮氨酸缺陷株），接種于 BioVector? 液體 LB 培養(yǎng)基中，37 攝氏度搖床培養(yǎng)至對數(shù)生長中期（每毫升約有 2.0乘以10的8次方 個活菌）。(Grow both the BioVector? Hfr donor and F-minus recipient into mid-log phase using clear LB broth.)

2. 取 1.0 mL 供體菌液與 9.0 mL 受體菌液混合于無菌大試管中（混合比例通常為 1比9，確保受體菌過量），輕輕混勻。(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. 立即將試管置于 37 攝氏度水浴或孵箱中靜置培養(yǎng)（0 rpm），正式開始接合記時。(Place the mixture statically inside a 37 degrees Celsius water bath; start timing the conjugation sequence.)

4. 根據(jù)基因圖譜測定需求，在接合開始后的第 5 分鐘、10 分鐘、15 分鐘、20 分鐘等設(shè)定時間點，分別吸取 0.5 mL 混合液，立刻投入冰預(yù)冷的無菌小管中，并使用渦旋振蕩器（Vortex）在最高速度下劇烈猛烈振蕩 60 秒。利用劇烈的機(jī)械剪切力強(qiáng)行成片折斷性菌毛，終止基因繼續(xù)轉(zhuǎn)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. 將劇烈振蕩后的樣品進(jìn)行適當(dāng)梯度稀釋，全量涂布于含有鏈霉素且缺失蘇氨酸/亮氨酸的 BioVector? M9 最小鹽選擇性瓊脂平板 上。(Perform serial dilutions and plate onto BioVector? M9 selective agar containing streptomycin but lacking leucine/threonine.)

6. 將平板倒置于 37 攝氏度孵箱培養(yǎng) 24 到 48 小時。計算各個時間點長出的重組子菌落數(shù)量，即可根據(jù)菌落首次出現(xiàn)的時間，換算出目標(biāo)基因在染色體基因組上的相對物理分鐘位置。(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 甘油菌凍存復(fù)蘇與長期保存 / Cryopreservation and Revitalization Workflow

1. 甘油冷凍管配制：在超凈臺內(nèi)，吸取 800微升 處于對數(shù)生長旺盛期的 BioVector? Hfr 液體菌液，加入到盛有 200微升 無菌無毒純甘油的冷凍管中（最終甘油體積分?jǐn)?shù)達(dá)到 20%），徹底顛倒搖勻。

2. 配制完成后，將冷凍管直接投入零下 80 攝氏度超低溫冰箱中，可穩(wěn)定保存 2 到 3 年；長期永久保存建議放置于液氮氣相環(huán)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. 凍存回收活化：從零下 80 攝氏度冰箱中取出凍存管，置于冰盒上微融。在超凈臺內(nèi)使用無菌接種環(huán)或槍頭，直接刮取冰霜表層少許菌體，在預(yù)熱的 BioVector? LB 固體平板 上進(jìn)行功能劃線，隨后將凍存管迅速放回深冷冰箱，嚴(yán)禁發(fā)生反復(fù)徹底凍融。(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. 將劃線平板置于 37 攝氏度孵箱培養(yǎng) 16 到 24 小時，即可獲得高活力的純系單菌落。

7 生物安全、純度控制與質(zhì)控規(guī)范 / Biosafety, Purity Controls and Quality Assays

• 生物安全級別 (Biosafety Level)：BSL-1。大腸桿菌 K12 譜系屬于國際公認(rèn)的非致病性安全實驗室常規(guī)工程菌，無人類感染和擴(kuò)散風(fēng)險。日常操作執(zhí)行基礎(chǔ)無菌規(guī)范，實驗殘液及平板在廢棄前需執(zhí)行 121 攝氏度高壓蒸汽滅菌 30 分鐘。(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 因子整合狀態(tài)定期抽檢（質(zhì)控紅線）/ Functional Recombination Quality Check：整合在宿主染色體上的 F 因子存在極低的自發(fā)自解離概率。如果自發(fā)解離并帶有宿主部分基因，則退化為 F一撇（F-prime）表型；如果完全解離則退化為普通 F加。若連續(xù)傳代或保存不當(dāng)，Hfr 菌株將徹底喪失高頻定向轉(zhuǎn)移整段染色體的表型。 嚴(yán)格質(zhì)控表現(xiàn)為：每隔 6 個月或每批量配制甘油庫時，必須抽樣一管與標(biāo)準(zhǔn)的已知缺陷型 F減 受體菌進(jìn)行交配對照測試。如果測試發(fā)現(xiàn)其在 15 分鐘內(nèi)無法驅(qū)動前位標(biāo)記基因發(fā)生高頻重組轉(zhuǎn)導(dǎo)（重組效率低于初始質(zhì)控基準(zhǔn)的一個數(shù)量級），則證實該批菌株已經(jīng)發(fā)生嚴(yán)重降解退化，必須全盤銷毀，并重新向原廠申請低傳代原始原始菌種進(jìn)行恢復(fù)。 (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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