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OverExpress C43 (DE3) pLysS Chemically Competent Cells (SOLOS) > 1 x 10e6 cfu/ug Add to Cart

Cat#: 60448-1-LU
Quantity: 12 transformations (12 x 50 ul)
Price: 344 €
Supplier: Lucigen
Shipping: Dry Ice
User Manual  

OverExpress Competent Cells - Express a wide variety of toxic proteins

• Selected for conferring tolerance to toxic proteins
• 4 strains: C41(DE3), C41(DE3)pLysS, C43(DE3) & C43(DE3)pLysS
• ComboPack available

E. coli BL21(DE3) strains, like Lucigen´s E. cloni EXPRESS Competent Cells provide reliable expression of many proteins cloned into T7 expression vectors (e.g., pET or Lucigen’s pSMART®-cDNA vectors). However, in some cases expression is minimal or not detectable because the recombinant protein, when expressed, is deleterious or lethal to these standard BL21 strains. Examples of such toxic proteins include many membrane proteins, some cytoplasmic proteins, and nucleases. Unfortunately, successful expression of one or more toxic proteins is often important to the experimental goal.

Lucigen´s OverExpress Electrocompetent and Chemically Competent Cells are E. coli strains that are effective in expressing toxic proteins from all classes of organisms, including eubacteria, yeasts, plants, viruses, and mammals. The effectiveness of these new strains in expressing toxic proteins has been validated in more than 350 publications.

The OverExpress strains contain genetic mutations phenotypically selected for conferring tolerance to toxic proteins. The strain C41(DE3) was derived from BL21(DE3). This strain has at least one uncharacterized mutation, which prevents cell death associated with expression of many recombinant toxic proteins. The strain C43(DE3) was derived from C41(DE3) by selecting for resistance to a different toxic protein and can express a different set of toxic proteins to C41(DE3). Figure 1 graphically illustrates the advantages of the OverExpress Competent Cells, compared to standard BL21(DE3) cells, in expressing toxic proteins.

Image
GFP and RFP expression in OverExpress Cells.
Green Fluorescent Protein (top) or Red Fluorescent Protein (bottom) expressed from a T7 promoter construct that was transformed into C41, BL21, or C43 competent cells spread on IPTG plates to induce protein expression.


Image
Comparison of OverExpress C41(DE3) and C43(DE3) cells with the parental strain BL21(DE3).
Transformation efficiency and expression of heterologous proteins were analyzed.

As in standard BL21(DE3) strains, OverExpress C41(DE3), C41(DE3)pLysS, C43(DE3), and C43 (DE3)pLysS are lysogens of lambdaDE3. These strains carry a chromosomal copy of the T7 RNA Polymerase gene under the control of the lacUV5 promoter. These strains are suitable for production of protein from target genes cloned into T7-driven expression vectors. OverExpress C41(DE3), C41(DE3) pLysS, C43(DE3), and C43(DE3) pLysS are also deficient in the lon and ompT proteases.

OverExpress C41(DE3)pLysS and C43(DE3)pLysS also carry a chloramphenicol-resistant plasmid that encodes T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase. Cells containing pLysS produce a small amount of T7 lysozyme. These strains are used to suppress basal expression of T7 RNA polymerase prior to induction, thus stabilizing recombinants encoding particularly toxic proteins.

Because there are no intrinsic antibiotic resistances (or plasmids) in either C41(DE3) or C43(DE3), the strains can be differentiated from each other and from BL21(DE3) by transformation with a plasmid verification vector, pAVD10. pAVD10 contains the uncF gene (encoding the beta-subunit of E. coli ATPase) under the control of the T7 promoter. This plasmid is lethal to BL21(DE3) and to induced C41(DE3), but it is tolerated by C43(DE3) regardless of induction. pAVD10 is provided with OverExpress Cells.

Which OverExpress cell strain should I use?
It is difficult to predict which of the four OverExpress strains – C41(DE3), C43(DE3), C41(DE3)pLysS, or C43(DE3)pLysS – will work best in expressing a given protein. We recommend initially using the OverExpress ComboPack™, which contains 3 reactions each of the four OverExpress competent cell strains, to determine which one is best for your application. The OverExpress strains are available as electrocompetent or chemically competent cells.

OverExpress Genotypes
OverExpress C41(DE3): F – ompT hsdSB (rB- mB-) gal dcm (DE3)
OverExpress C41(DE3)pLysS: F – ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CmR)
OverExpress C43(DE3): F – ompT hsdSB (rB- mB-) gal dcm (DE3)
OverExpress C43(DE3)pLysS: F – ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CmR)

Order Information
Each OverExpress Kit contains: the indicated OverExpress Electrocompetent or Chemically Competent Cells in SOLO packaging (1 transformation per tube), Expression Recovery Medium (lactose minus) (also available separately), pUC19 Positive Control Plasmid, pAVD10 Verification Plasmid, and complete protocols. ComboPacks contain 3 reactions each of C41(DE3), C43(DE3), C41(DE)pLysS, and C43(DE3)pLysS, as either electrocompetent of chemically competent cells, as indicated.

Related Links

Overview of Electroporation Competent Cells
Overview of Chemically Competent Cells
OverExpress References (incl. list of cloned genes)

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Technical Product Information Sheet
Product Brochure


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