無內(nèi)毒素大腸桿菌菌株及感受態(tài)細(xì)胞

-BioVector NTCC典型培養(yǎng)物保藏中心

產(chǎn)品描述

無內(nèi)毒素大腸桿菌細(xì)胞產(chǎn)生一種修飾的脂多糖（脂質(zhì)IVA），該物質(zhì)不會(huì)在人體細(xì)胞中觸發(fā)內(nèi)毒素反應(yīng)。無內(nèi)毒素大腸桿菌細(xì)胞缺乏用于激活人TLR4/MD-2的外膜激動(dòng)劑；因此，與野生型大腸桿菌細(xì)胞相比，無內(nèi)毒素大腸桿菌對hTLR4/MD-2信號(hào)通路的激活要低數(shù)個(gè)數(shù)量級(jí)。從無內(nèi)毒素大腸桿菌制備的異源蛋白基本上沒有內(nèi)毒素活性。經(jīng)過從無內(nèi)毒素大腸桿菌細(xì)胞進(jìn)行最低限度的純化后，獲得的蛋白質(zhì)或質(zhì)粒（可能含有脂質(zhì)IVA）可用于大多數(shù)應(yīng)用場景，而不會(huì)在人體細(xì)胞中引起內(nèi)毒素反應(yīng)。

在無內(nèi)毒素大腸桿菌細(xì)胞中，正常情況下為六酰化脂多糖的兩個(gè)次級(jí)?；湵粍h除，從而消除了其在真核細(xì)胞中內(nèi)毒素毒性的一個(gè)關(guān)鍵決定因素。脂多糖的六個(gè)酰基鏈?zhǔn)菑?fù)合物中的Toll樣受體4（TLR4）識(shí)別并與髓系分化因子2（MD-2）結(jié)合的觸發(fā)點(diǎn)，從而導(dǎo)致NF-?B的激活和促炎細(xì)胞因子的產(chǎn)生。這兩個(gè)次級(jí)?；湹娜笔?dǎo)致了脂質(zhì)IVA的產(chǎn)生，該物質(zhì)不會(huì)誘導(dǎo)活化的異源四聚體TLR4/MD-2復(fù)合物的形成，因此不會(huì)觸發(fā)內(nèi)毒素反應(yīng)。此外，寡糖鏈也被刪除，這使得從任何下游產(chǎn)物中去除所產(chǎn)生的脂質(zhì)IVA變得更加容易。

無內(nèi)毒素大腸桿菌K-12細(xì)胞是通過對K-12 endA- recA-菌株進(jìn)行七處基因缺失改造而得到的，這些改造將脂多糖修飾為脂質(zhì)IVA，同時(shí)一個(gè)額外的補(bǔ)償性突變（msbA52）使細(xì)胞能夠在脂多糖前體脂質(zhì)IVA存在下維持生存能力。對無內(nèi)毒素大腸桿菌細(xì)胞進(jìn)行的具體突變組合如下：

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

無內(nèi)毒素大腸桿菌K-12細(xì)胞旨在用于生產(chǎn)低內(nèi)毒素的質(zhì)粒DNA。對于初代克隆工作，BioVector建議使用標(biāo)準(zhǔn)的高效感受態(tài)細(xì)胞，例如E. cloni? 10G ELITE電擊感受態(tài)細(xì)胞。然后可以將質(zhì)粒從初代宿主轉(zhuǎn)移到無內(nèi)毒素大腸桿菌K-12中，以生產(chǎn)低內(nèi)毒素質(zhì)粒。

無內(nèi)毒素大腸桿菌K-12細(xì)胞的生長與菌落特征：

無內(nèi)毒素大腸桿菌K-12細(xì)胞的生長速度約為正常K-12 endA- recA-細(xì)胞的50%。用戶需注意，在涂布轉(zhuǎn)化子后的最初24小時(shí)內(nèi)，會(huì)看到非常小的菌落。BioVector建議在挑取菌落用于后續(xù)實(shí)驗(yàn)之前，將平板培養(yǎng)32-48小時(shí)（更多信息請參閱本手冊的轉(zhuǎn)化方案部分）?？赡苄枰L的培養(yǎng)時(shí)間或使用起始培養(yǎng)物來達(dá)到所需的細(xì)胞密度。

此外，用戶在涂布無內(nèi)毒素大腸桿菌細(xì)胞時(shí)可能會(huì)觀察到菌落大小存在一些差異。一小部分（<2%）菌落可能比一般菌落大。這些較大的菌落與平均大小的菌落具有相似的特性。

用戶應(yīng)采取預(yù)防措施以盡量減少污染，因?yàn)樵摼晟L速度較慢，可能會(huì)增加污染風(fēng)險(xiǎn)。

無內(nèi)毒素大腸桿菌細(xì)胞具有特殊設(shè)計(jì)的膜組成，與大多數(shù)大腸桿菌菌株相比，需要改良的培養(yǎng)基配方。請注意以下幾點(diǎn)：

• 無內(nèi)毒素大腸桿菌細(xì)胞對滲透壓敏感，需要在其生長培養(yǎng)基中添加1%的NaCl。我們強(qiáng)烈建議您使用高鹽LB-Miller培養(yǎng)基以獲得最佳生長效果。

• 我們建議不要使用LB-Lennox或Superbroth培養(yǎng)基培養(yǎng)無內(nèi)毒素大腸桿菌細(xì)胞。這些培養(yǎng)基會(huì)導(dǎo)致細(xì)胞生長緩慢且效果不佳。

• 不要在培養(yǎng)基中添加Mg2?和Ca2?。它們已被證明會(huì)抑制無內(nèi)毒素大腸桿菌細(xì)胞的生長。

• 無內(nèi)毒素大腸桿菌細(xì)胞在液體培養(yǎng)中傾向于聚集。我們建議您在測量OD600之前，先渦旋震蕩細(xì)胞懸液。

無內(nèi)毒素大腸桿菌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-?B 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