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

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