Optimized for cloning toxic or unstable DNA

• Stabilize toxic inserts in common cloning and expression vectors (pUC and pET-type vectors)
• Clone and maintain challenging sequences at reduced plasmid copy number, then induce to high copy number for DNA recovery
• Avoid T1 and T5 phage contamination with tonA mutation
• Choose chemically competent cells for general cloning or electrocompetent cells for demanding applications such as library generation

Applications

• Cloning of unstable DNA sequences or those expressing toxic proteins.

CopyCutter™ EPI400™ E. coli* cells were developed to significantly lower the copy number of a wide variety of common vectors so that you can more readily clone unstable DNA sequences. DNA that is unstable at high-copy number often codes for a protein that inhibits cell growth or contains AT- and GC-rich sequences or sequences with strong secondary structure (Fig. 2).¹

The CopyCutter EPI400 cell line was derived from Epicentre's high-transformation efficiency phage T1-resistant TransforMAX™ EC100™-T1^RE.coli strain by manipulating a gene that controls the copy number of vectors containing ColE1 or pMB1 origins of replication (e.g., pUC- and pET-type vectors). This constitutively expressed gene, pcnB (plasmid copy number), was deleted from the TransforMAX EC100 strain and replaced with a modified pcnB gene that is linked to an inducible promoter, creating the CopyCutter EPI400 strain.

The copy number of ColE1-type vectors in the CopyCutter EPI400 strain compared to the parental TransforMAX EC100 strain is approximately 4- to 25-fold lower, depending on the vector. Moreover, a short incubation in the presence of the CopyCutter Induction Solution can increase the copy number of the vector to improve plasmid yields (Fig. 3).

Figure 1. Copy-number of ColE1-type plasmids is lowered up to 25-fold in CopyCutter™ EPI400™ E. coli cells. Lanes C, TransforMax™ EC100™ cells; Lanes U and I, uninduced or induced CopyCutter EPI400 cells. DNA extracts from the same number of lysed cells (based on OD₆₀₀) were loaded per lane.

Figure 2. DNA inserts encoding toxic gene products were successfully cloned into high-copy-number vectors using CopyCutter™ EPI400™ E. coli cells. After sequencing, the full-length acpP clones in TransforMAX™ EC100™ cells were found to contain multiple point mutations.

Figure 3. Uninduced CopyCutter™ EPI400™ E. coli cells containing a regB clone (lane U) are induced to higher-copy number (lane I) using the CopyCutter Induction Solution. Crude extracts of plasmid DNA were prepared from cells grown in selective media and analyzed by agarose gel electrophoresis. Approximately the same number of lysed cells (based on A₆₀₀) were loaded per lane.

Benefits

• Maintain clones at low-copy number, then induce to higher copy number for improved plasmid yield.
• High transformation efficiency with clones of all sizes.
• tonA for resistance to bacteriophages T1 and T5.
• lacZΔM15 for blue/white screening of recombinants.
• Restriction minus [mcrA, Δ(mrr-hsdRMS-mcrBC)] genotype enables efficient cloning of methylated DNA.
• Endonuclease minus (endA1) to ensure high yields of DNA.
• Recombination minus (recA1) for greater stability of large cloned inserts.

Genotype

F^- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ^- rpsL (Str^R) nupG trfA tonA pcnB4 dhfr

CopyCutter EPI400 Electrocompetent E. coli

• Transformation efficiency of >1 x 10¹⁰ cfu/µg of pUC19.

CopyCutter EPI400 Chemically Competent E. coli

• Transformation efficiency of >1 x 10⁷ cfu/µg of pUC19.

Reference

1. Haskins, D. (2004) Epicentre Forum 11(5), 6.

*Covered by issued and/or pending patents.