BioVector? SHuffle? T7 Express 大腸桿菌表達表達菌株——產(chǎn)品技術(shù)說明書

1 菌株基本信息與工程核心背景

產(chǎn)品名稱：BioVector? SHuffle? T7 Express 表達菌株

常用別名：SHuffle T7 Express Competent E. coli，SHuffle T7 表達株

生物學(xué)分類：細菌界 Bacteria / 變形菌門 Proteobacteria / 腸桿菌科 Enterobacteriaceae / 腸桿菌屬 Escherichia

細胞衍生背景：該菌株衍生自 Escherichia coli B 表達系（具有天然缺乏 Lon 和 OmpT 蛋白酶的優(yōu)勢），通過重組工程對細胞內(nèi)的氧化還原代謝通路進行了根本性的基因改造。

核心科研與工業(yè)價值：SHuffle? T7 Express 是專為在大腸桿菌細胞質(zhì)內(nèi)高效表達“富含二硫鍵、易形成包涵體”的復(fù)雜重組蛋白而設(shè)計的革命性底盤。 它克服了傳統(tǒng)大腸桿菌細胞質(zhì)高度還原性的限制，廣泛應(yīng)用于單克隆抗體片段（scFv、Fab）、含有大量半胱氨酸的細胞因子、毒素蛋白、受體胞外域以及需要精密正確折疊的工業(yè)酶類的體外高活性表達。

2 核心基因型特征與三大重組折疊機制

SHuffle? T7 Express 菌株的核心基因型（以特定改良型為準）蘊含著其三大核心蛋白質(zhì)折疊優(yōu)化系統(tǒng)：

• $\Delta gor \Delta trxB$ 雙基因缺失（打破細胞質(zhì)還原環(huán)境）：細胞內(nèi)控制谷胱甘肽還原酶（$gor$）和硫氧還蛋白還原酶（$trxB$）的基因被完全敲除。這使得細胞質(zhì)由原本的強還原性自發(fā)轉(zhuǎn)變?yōu)?strong data-path-to-node="4,0,0" data-index-in-node="100" style="line-height:1.15 !important;margin-top:0px !important;">氧化性環(huán)境，允許進入細胞質(zhì)的半胱氨酸巰基（-SH）能夠自發(fā)氧化形成二硫鍵（-S-S-）。

• 染色體集成表達 DsbC 巰基/二硫鍵異構(gòu)酶（修正錯誤折疊）：該菌株在染色體上穩(wěn)定集成并高表達原本屬于周質(zhì)空間的二硫鍵異構(gòu)酶 DsbC（去除了周質(zhì)定位信號，使其駐留細胞質(zhì)）。DsbC 在細胞質(zhì)中扮演“折疊監(jiān)視器”，能夠高效催化并斷開因氧化環(huán)境錯誤連接的二硫鍵，并協(xié)助其重新排列組合成正確的天然構(gòu)象，從源頭上減少包涵體的產(chǎn)生。

• 染色體集成 T7 RNA 聚合酶（高效轉(zhuǎn)錄系統(tǒng)）：其基因組中集成了受 lac 啟動子控制的 T7 RNA 聚合酶（類似于 BL21(DE3) 的工作原理）。這使得它完美兼容所有基于 T7 啟動子的表達載體（如 pET 系列、pGEX 系列等），只需加入 IPTG 即可啟動強效的靶蛋白轉(zhuǎn)錄與翻譯。

• 抗生素抗性譜：該菌株常染色體天然攜帶壯觀霉素抗性（Spectinomycin-resistant, $Spec^R$），在日常菌種維持時需注意其抗性背景，避免與相同抗性的質(zhì)粒沖突。

3 專用生長培養(yǎng)基與誘導(dǎo)配方規(guī)范

SHuffle? T7 Express 的生長速度和質(zhì)粒穩(wěn)定性與培養(yǎng)基質(zhì)量密切相關(guān)。

A 標(biāo)準 LB 培養(yǎng)基/瓊脂平板配方（用于日常常規(guī)維持與轉(zhuǎn)化克?。?/p>

• 基礎(chǔ)成分：胰蛋白胨（Tryptone）10.0 g，酵母提取物（Yeast Extract）5.0 g，氯化鈉 NaCl 10.0 g。

• 瓊脂補劑（僅限平板固化）：瓊脂粉 15.0 g。

• 溶劑：蒸餾水或去離子水 1000 mL。

• 滅菌規(guī)范：121 ℃ 高壓蒸汽滅菌 15 分鐘。

B 高密度表達培養(yǎng)基選擇（推薦）

• TB（Terrific Broth）培養(yǎng)基：相比于 LB，TB 培養(yǎng)基具有更強的緩沖能力和更高的營養(yǎng)濃度（富含甘油作為碳源），能夠支持 SHuffle? 菌株生長到更高的菌體密度，從而顯著提升單位體積的靶蛋白產(chǎn)率。

C 菌株長期凍存保護液

• 標(biāo)準配方：無菌 LB 肉湯 80% + 細胞/細菌級純甘油 20%。與對數(shù)生長期的菌液充分混合后，分裝保存于深度低溫環(huán)境中。

4 物理環(huán)境控制參數(shù)

• 培養(yǎng)溫度（常規(guī)擴增）：37.0 ℃（最適分裂溫度）。

• 誘導(dǎo)折疊溫度（紅線紅字）：20.0 ℃ 至 30.0 ℃（低溫誘導(dǎo)）。雖然該菌株支持 37 ℃ 生長，但在加入 IPTG 誘導(dǎo)靶蛋白表達時，強烈建議將溫度降低至 16 ℃ - 25 ℃ 展開暗誘導(dǎo)。低溫能夠顯著減緩核糖體的翻譯速度，給予 DsbC 異構(gòu)酶充分的時間去糾正和穿鑿二硫鍵，最大化提升可溶性活性蛋白的比例。

• 氣相環(huán)境：常規(guī)常壓無菌空氣，需氧震蕩培養(yǎng)（建議搖床轉(zhuǎn)速 220-250 rpm，保證充足溶氧）。

5 感受態(tài)細胞轉(zhuǎn)化與誘導(dǎo)表達操作規(guī)范

標(biāo)準熱激轉(zhuǎn)化步驟：

1. 從 -80 ℃ 超低溫冰箱中取出一管 SHuffle? T7 Express 感受態(tài)細胞（通常 50 uL/管），快速置于冰上融化（約 2-5 分鐘）。

2. 向融化的感受態(tài)細胞中加入 1 到 3 uL 構(gòu)建好的高純度重組質(zhì)粒（如 pET-28a-Target），用手指輕彈管底混勻，切勿使用移液槍吹打。

3. 置于冰上靜置冰浴 30 分鐘。

4. 將離心管快速投入 42.0 ℃ 恒溫水浴箱中，準確熱擊 45 秒，隨后立刻秒速移回冰上，靜置冰浴 2 分鐘（切勿搖動）。

5. 向管中加入 400-500 uL 不含抗生素的預(yù)熱 SOC 或 LB 液體培養(yǎng)基，置于 37 ℃ 搖床中，以 200 rpm 復(fù)蘇培養(yǎng) 60 分鐘。

6. 取 100 uL 復(fù)蘇菌液涂布于含有靶向質(zhì)?？剐裕ㄈ缈敲顾?Kan 或氨芐青霉素 Amp）的 LB 瓊脂平板上。倒置放入 37 ℃ 孵箱培養(yǎng) 16-20 小時直至長出清晰的小單菌落。

標(biāo)準重組蛋白誘導(dǎo)流程：

1. 挑取平板上的單菌落，接種至 5 mL 含相應(yīng)抗生素的 LB 或 TB 肉湯中，37 ℃ 220 rpm 培養(yǎng)過夜作為種子液。

2. 按 1:100 的比例將種子液轉(zhuǎn)接至大體積表達培養(yǎng)基中，37 ℃ 震蕩培養(yǎng)直至光吸收值 $OD_{600}$ 達到 0.4 到 0.6 之間。

3. 關(guān)鍵降溫階段：將培養(yǎng)瓶從 37 ℃ 搖床中取出，置于室溫或預(yù)冷搖床中，使其物理降溫至 20 ℃ 保持 15-20 分鐘。

4. 加入終濃度為 0.1 到 1.0 mM 的 IPTG（異丙基硫代半乳糖苷）啟動誘導(dǎo)。

5. 將搖床參數(shù)調(diào)整為 20 ℃（或 16 ℃ 保持更長時間），200 rpm 持續(xù)誘導(dǎo)表達 12 到 24 小時。

6. 離心收集菌體，通過超聲破碎法或溫和溶菌酶裂解法收獲細胞質(zhì)總蛋白，進行 SDS-PAGE 以及可溶性/活性分析。

6 長期封存與質(zhì)控規(guī)范

• 生物安全級別：BSL-1（常規(guī)實驗室安全防護）。屬于安全、無致病性的實驗室工程安全株。所有廢棄培養(yǎng)物按常規(guī)高壓滅菌程序處理即可。

• 深度超低溫保存：甘油凍存管必須永久存放于 -80 ℃ 超低溫冰箱或液氮中。由于缺失了 $gor$ 和 $trxB$，該菌株在非保護環(huán)境下對環(huán)境壓力的耐受度低于普通 BL21 菌株，嚴禁在 -20 ℃ 或 4 ℃ 下長期擱置菌種。

• 蛋白質(zhì)可溶性抽檢質(zhì)控：在批量使用該表達系統(tǒng)前，建議采用帶有特定二硫鍵標(biāo)記的對照質(zhì)粒（如重組綠色熒光蛋白 tGFP，折疊錯誤時不發(fā)光）進行轉(zhuǎn)化測試。通過測定發(fā)光效率或可溶性上清中的蛋白收率，確保該批次細胞的 DsbC 輔助折疊活性未發(fā)生基因退化或自發(fā)突變丟失。

BioVector? SHuffle? T7 Express Competent E. coli Expression Strain Product Datasheet

1 Strain and Engineering Identification General Information

• Product Name: BioVector? SHuffle? T7 Express Expression Strain

• Synonyms: SHuffle T7 Express Competent E. coli, SHuffle T7 Expression Host

• Biological Taxonomy: Kingdom Bacteria / Phylum Proteobacteria / Family Enterobacteriaceae / Genus Escherichia

• Lineage Context: This customized host is derived from the Escherichia coli B strain background (inherently deficient in Lon and OmpT protease systems) and has undergone core genomic rewiring of its intracellular redox metabolic pathways.

• Core Research & Industrial Value: SHuffle? T7 Express is a revolutionary bacterial chassis engineered specifically for the high-yield expression of complex, disulfide-bond-rich recombinant proteins that typically form insoluble inclusion bodies in standard E. coli. By converting the highly reducing cytoplasmic baseline into a functional oxidative workspace, it serves as an international gold standard for producing monoclonal antibody fragments (scFv, Fab), cysteine-dense cytokines, mammalian receptors, and properly folded industrial enzymes within the cytoplasm.

2 Core Genotypic Hallmarks & Triple Folding Optimization Mechanisms

The comprehensive genomic configuration of the SHuffle? T7 Express strain drives three independent, synergistic protein-folding engines:

• $\Delta gor \Delta trxb$ Double Deletion (Eliminating Cytoplasmic Reducing Power): The simultaneous knockout of the glutathione reductase ($gor$) and thioredoxin reductase ($trxB$) genes prevents the reduction of cysteines. This converts the cytoplasm into a stably oxidative environment, allowing cysteine sulfhydryl groups (-SH) to actively mature into disulfide bonds (-S-S-) directly upon translation.

• Chromosomally Integrated DsbC Disulfide Isomerase (Correcting Misfolded Intermediates): The strain features a permanent genomic integration of the disulfide isomerase DsbC, stripped of its periplasmic leader sequence so it remains active within the cytoplasm. DsbC acts as an intracellular chaperone that scans newly formed proteins, cleaving non-native or mismatched disulfide linkages and isomerizing them into their correct biological architecture, significantly lowering inclusion body formation.

• Chromosomal T7 RNA Polymerase (High-Efficiency Transcription): Harbors a lysogenic copy of the T7 RNA polymerase gene controlled by the inducible lac promoter (equivalent to the expression dynamics of a BL21(DE3) setup). This allows seamless compatibility with any T7-driven vector systems (such as pET or pGEX arrays) via standard IPTG induction.

• Antibiotic Resistance Profile: Naturally carries a chromosomally encoded Spectinomycin resistance ($Spec^R$) cassette. Researchers must note this background selection marker to ensure it does not conflict with incoming experimental plasmid resistance.

3 Dedicated Growth Media and Induction Formulations

The doubling speed and plasmid retention profile of SHuffle? T7 Express rely heavily on premium nutrient selection.

A Standard LB Medium / Agar Plates (For Clone Maintenance and Transformation Verification)

• Basal Macro-Nutrients: Tryptone 10.0 g, Yeast Extract 5.0 g, Sodium Chloride (NaCl) 10.0 g.

• Solidifying Matrix (For plates only): Agar powder 15.0 g.

• Solvent Matrix: Distilled or Deionized Water 1000 mL.

• Sterilization Cycle: Autoclave at 121 ℃ for 15 minutes.

B High-Density Expression Media Recommendations

• TB (Terrific Broth) Medium: Strongly recommended over standard LB for large-scale production runs. TB possesses enhanced buffering capacities and increased nutrient density (utilizing glycerol as a primary carbon feed), enabling SHuffle? cultures to achieve significantly higher optical densities ($OD_{600}$) and superior volumetric protein target yields.

C Cryopreservation Archiving Fluid

• Glycerol Stock Formula: 80% Sterile LB Broth Matrix + 20% Analytical/Bacterial Grade Pure Glycerol. Blend uniformly with active log-phase cultures prior to long-term storage under ultra-low freezing configurations.

4 Controlled Environmental Parameters

• Incubation Temperature (Growth): 37.0 ℃ (Optimum operating index for rapid binary fission).

• Induction Folding Temperature (Critical Threshold): 20.0 ℃ to 30.0 ℃ (Low-Temperature Induction Window). While the biomass expands efficiently at 37 ℃, it is imperative to drop the workstation temperature to 16 ℃ - 25 ℃ during active IPTG induction. Lowering thermal parameters slows down ribosomally mediated translation kinetics, granting the cytoplasmic DsbC machinery adequate time to scan, dock, and isomerize complex disulfide configurations, maximizing the soluble target ratio.

• Gas Phase Configuration: Standard atmospheric oxygen environment with active agitating aeration (Orbital shaker set to 220-250 rpm to avoid hypoxia).

5 Transformation and Recombinant Expression Protocols

Standard Heat-Shock Transformation Protocol:

1. Retrieve a 50 uL aliquot of frozen SHuffle? T7 Express competent cells from -80 ℃ storage and immediately place on ice to thaw gently (approx 2-5 minutes).

2. Introduce 1 to 3 uL of high-purity recombinant plasmid DNA (e.g., pET-28a construct) directly into the thawed matrix. Mix gently by tapping the bottom of the tube with a finger; never pipette or vortex the fragile cells.

3. Incubate the cellular mixture on ice for exactly 30 minutes.

4. Transfer the tube instantly into a 42.0 ℃ calibrated water bath for a strict 45-second heat shock. Transfer immediately back onto ice for 2 minutes without agitating.

5. Add 400-500 uL of pre-warmed, antibiotic-free SOC or LB media to the tube, and incubate in a shaking incubator at 37 ℃, 200 rpm for 60 minutes to recover phenotype expression.

6. Spread a 100 uL aliquot of the recovered pool onto an LB agar plate supplemented with the target plasmid antibiotic (e.g., Kanamycin Kan or Ampicillin Amp). Invert the plates and incubate at 37 ℃ for 16-20 hours until single colonies emerge.

Standard Inducible Protein Expression Pipeline:

1. Inoculate a single isolated colony from the transformation plate into 5 mL of LB or TB broth containing appropriate selection antibiotics. Shake over-night at 37 ℃ at 220 rpm to establish a starter seed stock.

2. Inoculate the starter culture at a 1:100 dilution ratio into the final production volume. Shake at 37 ℃ at 220 rpm until the cell density reaches an $OD_{600}$ index of 0.4 to 0.6.

3. Thermal Equilibrium Phase: Remove the production vessels from the 37 ℃ environment and transition them to a pre-cooled workspace calibrated at 20 ℃ for 15-20 minutes to bring the liquid matrix to the correct induction temperature.

4. Introduce IPTG (Isopropyl $\beta$-D-1-thiogalactopyranoside) to a final operational concentration of 0.1 to 1.0 mM.

5. Operate the shaker platform at 20 ℃ (or 16 ℃ for longer runs) at 200 rpm for a continuous induction window of 12 to 24 hours.

6. Pelleting out the biomass via centrifugation, and disrupt the cell walls utilizing ultrasonic sonication or mild lysozyme enzymatic digestion to isolate the cytoplasmic soluble fraction for downstream SDS-PAGE or functional assay validation.

6 Long-Term Preservation and Quality Control Metrics

• Biosafety Classification: BSL-1 (Standard laboratory safety parameters). Represents a non-pathogenic, safe laboratory cloning and expression host. Discard spent matrices following routine institutional autoclaving disinfection runs.

• Ultra-Low Freeze Archiving: Glycerol vials must reside permanently at -80 ℃ or within liquid nitrogen repositories. Due to the deletion of cellular pathways ($gor$/$trxB$), this host exhibits reduced fitness to environmental fluxes compared to wild-type BL21; keeping liquid stocks at 4 ℃ or -20 ℃ for extended intervals will lead to sharp viability drops.

• Folding System Validation QC: Periodically evaluate the cytoplasmic folding integrity by transforming a control plasmid encoding a disulfide-dependent reporter protein (such as a recombinant green fluorescent protein tGFP variant that requires proper disulfide loops to fluoresce). Quantifying the specific fluorescence output or measuring soluble yield against a baseline BL21 control verifies that the genomic DsbC and pathway knockouts remain intact without phenotypic drifting.

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