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Cloning, cDNA
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CopyCutterTM, CopyControlTM

Gene and PCR Cloning Kit

CopyCutter™ EPI400™ Electrocompetent E. coli
CopyCutter™ EPI400™ Chemically Competent E. coli

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™-T1R E.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).

g_copycutter_fig1.jpg
 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.

i_copycutter_dnainserts.gif
g_copycutter_uninduced.jpg
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. colicells 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.
  • tonAfor resistance to bacteriophages T1 and T5.
  • lacZΔM15for 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 (StrR) 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.



CopyControl™ cDNA, Gene and PCR Cloning Kits

Applications
  • Efficient cloning of any insert up to 15 kb, including those that may be unstable or encode proteins that are toxic to the E. coli host cells.
Up to ~25% more cDNA clones can be obtained in a library if the cDNA clones are present in the host cell at only about one copy per cell (P. Carninci and W. Szybalski, personal communication), presumably due to reduced detrimental effects on host cell metabolism and lower toxicity of expressed clone sequences and gene products. The CopyControl™ cDNA, Gene, and PCR Cloning Kits* provide a rapid and improved method (Fig. 1) for cloning any blunt-end DNA fragments (e.g., genomic DNA, PCR products, or cDNA) at single-copy number, with the capability to induce to high-copy numbers as needed (Fig. 2). The CopyControl pCC1™ (Blunt) Vector provided in the kits contains both the E. coli F-factor single-copy origin of replication (oriII) and an inducible high-copy oriV. Replication does not occur using the oriV unless and until the trfA gene in the chromosome of TransforMax™ EPI300™ cells (which is under the control of a tightly regulated promoter) is induced. Thus, CopyControl cDNA, PCR, or gene clones can be grown at single copy to ensure insert stability and successful cloning of sequences that are toxic to the host cell. Then, any desired clone can be induced up to 200 copies per cell for high yields of DNA for all downstream applications.

Benefits
  • Obtain more cDNA or PCR clones and more complete clone libraries (Table 1).
  • Obtain fewer mutated clones. Clones having mutations in genes that are toxic to the host cell are selectively favored at high-copy number.
  • Screen clones for insert size in 1 hour or less without the need for overnight cultures or restriction endonuclease digests (Fig. 3).

i_copycontrol_bluntendcloning.gif
Figure 1 . Efficient, blunt-end cloning of inserts up to 15 kb and screening of clones can be completed in 24 hours using the CopyControl™ cDNA, Gene, and PCR Cloning Kit. Clones can be kept at one copy per cell for clone stability and then, whenever desired, any clone can be induced to high-copy number (up to 200 copies per cell) for high yields of DNA.

스크린샷 2013-06-25 오전 11.09.15.png
 Table 1. PCR product cloning results with the CopyControl™ System.

g_copycontrol_singlecopyclones.jpg
 Figure 2. Single-copy CopyControl™ clones can be induced to 200 copies per cell. DNA from an equal number of uninduced (–) cells (lane 1) and undiluted induced (+) cells (lane 5) containing a 10-kb CopyControl PCR clone was analyzed by electrophoresis in a 1% agarose gel. Copy number in induced cells was estimated by visually comparing the staining intensity of the diluted induced sample with the uninduced sample. DNA from induced cells was diluted 1:200 (lane 2), 1:100 (lane 3), and 1:50 (lane 4).
g_copycontrol_clonescreening.jpg
 Figure 3. Screening of clones from a 5-kb PCR product. The PCR product was cloned using the CopyControl™ cDNA, Gene, and PCR Cloning Kit. Very small portions from nine white colonies were screened using the kit, confirming the presence of the 5-kb insert. Lane M, DNA size ladder; lane 1, 8-kb supercoiled DNA marker; lanes 2-10, randomly chosen clones. Total time for colony screening, including gel electrophoresis, was approximately 60 minutes.

*Covered by issued and/or pending patents.
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