Endotoxin-Free E.coli strain and competent cells无内毒素大肠杆菌菌株及感受态细胞-BioVector NTCC质粒载体菌种细胞基因保藏中心

无内毒素大肠杆菌菌株及感受态细胞

-BioVector NTCC典型培养物保藏中心

产品描述

无内毒素大肠杆菌细胞产生一种修饰的脂多糖（脂质IVA），该物质不会在人体细胞中触发内毒素反应。无内毒素大肠杆菌细胞缺乏用于激活人TLR4/MD-2的外膜激动剂；因此，与野生型大肠杆菌细胞相比，无内毒素大肠杆菌对hTLR4/MD-2信号通路的激活要低数个数量级。从无内毒素大肠杆菌制备的异源蛋白基本上没有内毒素活性。经过从无内毒素大肠杆菌细胞进行最低限度的纯化后，获得的蛋白质或质粒（可能含有脂质IVA）可用于大多数应用场景，而不会在人体细胞中引起内毒素反应。

在无内毒素大肠杆菌细胞中，正常情况下为六酰化脂多糖的两个次级酰基链被删除，从而消除了其在真核细胞中内毒素毒性的一个关键决定因素。脂多糖的六个酰基链是复合物中的Toll样受体4（TLR4）识别并与髓系分化因子2（MD-2）结合的触发点，从而导致NF-ƙB的激活和促炎细胞因子的产生。这两个次级酰基链的缺失导致了脂质IVA的产生，该物质不会诱导活化的异源四聚体TLR4/MD-2复合物的形成，因此不会触发内毒素反应。此外，寡糖链也被删除，这使得从任何下游产物中去除所产生的脂质IVA变得更加容易。

无内毒素大肠杆菌K-12细胞是通过对K-12 endA- recA-菌株进行七处基因缺失改造而得到的，这些改造将脂多糖修饰为脂质IVA，同时一个额外的补偿性突变（msbA52）使细胞能够在脂多糖前体脂质IVA存在下维持生存能力。对无内毒素大肠杆菌细胞进行的具体突变组合如下：

msbA52 ∆gutQ ∆kdsD ∆lpxL ∆lpxM ∆pagP ∆lpxP ∆eptA

无内毒素大肠杆菌K-12细胞旨在用于生产低内毒素的质粒DNA。对于初代克隆工作，BioVector建议使用标准的高效感受态细胞，例如E. cloni® 10G ELITE电击感受态细胞。然后可以将质粒从初代宿主转移到无内毒素大肠杆菌K-12中，以生产低内毒素质粒。

无内毒素大肠杆菌K-12细胞的生长与菌落特征：

无内毒素大肠杆菌K-12细胞的生长速度约为正常K-12 endA- recA-细胞的50%。用户需注意，在涂布转化子后的最初24小时内，会看到非常小的菌落。BioVector建议在挑取菌落用于后续实验之前，将平板培养32-48小时（更多信息请参阅本手册的转化方案部分）。可能需要更长的培养时间或使用起始培养物来达到所需的细胞密度。

此外，用户在涂布无内毒素大肠杆菌细胞时可能会观察到菌落大小存在一些差异。一小部分（<2%）菌落可能比一般菌落大。这些较大的菌落与平均大小的菌落具有相似的特性。

用户应采取预防措施以尽量减少污染，因为该菌株生长速度较慢，可能会增加污染风险。

无内毒素大肠杆菌细胞具有特殊设计的膜组成，与大多数大肠杆菌菌株相比，需要改良的培养基配方。请注意以下几点：

• 无内毒素大肠杆菌细胞对渗透压敏感，需要在其生长培养基中添加1%的NaCl。我们强烈建议您使用高盐LB-Miller培养基以获得最佳生长效果。

• 我们建议不要使用LB-Lennox或Superbroth培养基培养无内毒素大肠杆菌细胞。这些培养基会导致细胞生长缓慢且效果不佳。

• 不要在培养基中添加Mg²⁺和Ca²⁺。它们已被证明会抑制无内毒素大肠杆菌细胞的生长。

• 无内毒素大肠杆菌细胞在液体培养中倾向于聚集。我们建议您在测量OD600之前，先涡旋震荡细胞悬液。

无内毒素大肠杆菌K-12基因型

F– λ– ∆endA– ∆recA– frr181 msbA52 ∆gutQ ∆kdsD ∆lpxL ∆lpxM ∆pagP lpxP ∆eptA

Endotoxin-Free E.coli strain and competent cells

-BioVector NTCC Inc.

Description

Endotoxin-Free E.coli cells produce a modified LPS (Lipid IVA)that does not trigger the endotoxic response in human cells. Endotoxin-FreeE.coli cells lack outer membrane agonists for hTLR4/MD-2 activation; therefore,activation of hTLR4/MD-2 signaling by Endotoxin-Free E.coli is several ordersof magnitude lower as compared with E. coli wild-type cells.Heterologous proteins prepared from Endotoxin-Free E.coli are virtually free ofendotoxic activity. After minimal purification from Endotoxin-Free E.colicells, proteins or plasmids (which may contain Lipid IVA)can be used in most applications without eliciting an endotoxic response inhuman cells.

In Endotoxin-Free E.coli cells, two ofthe secondary acyl chains of the normally hexa-acylated LPS have been deleted,eliminating a key determinant of endotoxicity in eukaryotic cells. The six acylchains of the LPS are the trigger which is recognized by the Toll-like receptor4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causingactivation of NF-ƙB andproduction of proinflammatory cytokines. The deletion of the two secondary acylchains results in lipid IVA,which does not induce formation of the activated heterotetrameric TLR4/MD-2complex and thus does not trigger the endotoxic response. In addition, theoligosaccharide chain is deleted, making it easier to remove the resultinglipid IVAfromany downstream product.

Endotoxin-Free E.coli K-12cells were derived from K-12 endA- recA- by making seven geneticdeletions that modify LPS to Lipid IVA, while oneadditional compensating mutation (msbA52) enables the cells to maintainviability in the presence of the LPS precursor lipid IVA.The specific set of mutations made to Endotoxin-Free E.coli cells is asfollows:

msbA52gutQkdsD lpxL lpxM pagPlpxP eptA

Endotoxin-Free E.coli K-12cells are intended to be used for production of low endotoxin plasmid DNA. Forprimary cloning work, BioVector recommends the use of a standardhigh-efficiency competent cell such as the E. cloni® 10GELITE Electrocompetent cells. Plasmids can then be transferred from the primaryhost to Endotoxin-Free E.coli K-12 to produce low-endotoxin plasmid.

Growth and ColonyCharacteristics of Endotoxin-Free E.coli K-12 Cells:

Endotoxin-Free E.coli K-12cells grow at approximately 50% of the rate of normal K-12 endA- recA-cells. Users should expect to see very small colonies for the first 24hours after plating transformants. BioVectorrecommends incubating plates for 32-48 hours before picking colonies for futureexperiments (see Transformation Protocol section of this manual for moreinformation). Longer growth times or a starter culture may be necessary toreach desired cell densities.

In addition, users may observe somevariation in colony size when plating Endotoxin-Free E.coli cells. A smallportion (<2%) of colonies may be larger than the general population. Theselarger colonies have similar properties to the average size colonies.

Usersshould take precautions to minimize contamination, as the slower growth rate ofthis strain may increase the risk of contamination.

Endotoxin-FreeE.coli cells have a specially engineered membrane composition and require amodified medium formulation compared to most E. coli strains. Considerthe following:

Endotoxin-FreeE.coli cells are osmosensitive and require 1% NaCl in their growthmedium. We strongly recommend you use high salt LB-Miller Medium toachieve optimal growth.

Wedo not recommend growing Endotoxin-Free E.coli cells in LB-Lennox or Superbroth medium. These media have resulted in slow and suboptimal cell growth.

Donot includeMg2+andCa2+inyour medium. They have been shown to inhibit growth of Endotoxin-Free E.colicells.

Endotoxin-FreeE.coli cells tend to aggregate when grown in liquid culture. We suggest thatyou vortex the cell solution before measuring OD600.

Endotoxin-FreeE.coli K-12 Genotype

F– λ– endA– recA– frr181msbA52 gutQ kdsD lpxL lpxMpagP lpxP eptA