Ultra-high competency—outperforming competitors’ cells

Stellar Competent Cells are an E. coli HST08 strain that lacks the gene group (mcrA, mrr – hsdRMS - mcrBC) responsible for cleaving foreign methylated DNAs. The cells are also an F– strain, which allows the use of BAC and fosmid vectors. In a transformation using a pUC plasmid, Stellar cells allow blue-white screening of recombinants with X-gal, based on β-galactosidase α-complementation. These cells display an excellent transformation potential even for large DNA fragments, making it easy to produce clones or libraries of fragments up to 20 kb.

A comparison of transformation efficiencies using purified plasmids

Transformation efficiencies of purified plasmids were compared using Stellar Chemically Competent Cells, DH5α competent cells, and DH10B competent cells. Their respective guaranteed transformation efficiencies are >1.0×10⁸, >1.0×10⁸ and >1.0×10⁹ colony forming units (CFU) per μg pUC19 DNA.

For each of the plasmid DNA sizes tested, Stellar Chemically Competent Cells provided transformation efficiencies equivalent to or better than those obtained using DH5α or DH10B competent cells (Figure 1). The high efficiency of Stellar Competent Cells was especially noticeable in the transformation of 10 kb and 20 kb plasmids. There, Stellar Competent Cells yielded approximately two fold more colonies than were obtained using DH5α and DH10B.

Figure 1. Comparison of transformation efficiencies using purified plasmids. Using DNA plasmids of 2 kb (100 pg), 10 kb (1 ng), and 20 kb (1 ng), each strain of competent cells was transformed and plated on ampicillin-containing LB agar plates. Transformation efficacies were determined based on the colony counts obtained.

A comparison of transformation efficiencies using ligation reaction mixtures

Transformation efficiencies of ligation reactions were compared using the same panel of competent cells as in Figure 1. The two ligation reactions were insertion of either a 2 kb fragment or a 20 kb fragment into the pUC118 vector.

Stellar Chemically Competent Cells provided the highest transformation efficiencies for all the ligation reaction mixtures tested (Figure 2). The difference was particularly prominent for cloning 20 kb fragments. This demonstrates the exceptional transformation efficiency of Stellar cells compared to DH5α and DH10B in cloning large fragments. The high transformation efficiency of Stellar Competent Cells not only improves ordinary cloning, but also increases the percentage of large fragments in cDNA and genomic libraries.

Figure 2. Comparison of transformation efficiencies using ligation reaction mixtures. Ligation reactions were set up with either 100 ng of a 2 kb, Hind III-cut DNA fragment + 50 ng of Hind III/BAP-cut pUC118 vector, or 75 ng of a 20 kb, Hind III-cut DNA fragment + 25 ng of Hind III/BAP-cut pUC118 vector. All reactions were performed at 16˚C for 6 hours using the DNA Ligation Kit, Mighty Mix or DNA Ligation Kit, LONG. Each strain of competent cells was transformed using portions of these reaction mixtures and plated on ampicillin containing LB agar (+ X-gal). Transformation efficiencies were determined based on the white colony counts obtained.

A comparison of colony growth rate on agar after transformation

When the 2 kb plasmid was transformed into each strain, there was no obvious difference in the colony growth rates between Stellar and DH10B competent cells (Figure 3, Panel A). However, Stellar colonies transformed with larger sized 10 kb plasmid clearly showed a faster growth rate than DH10B colonies (Figure 3, Panel B). It has frequently been observed that the transformation of a large fragment slows the growth of some E. coli strains. The Stellar strain, nevertheless, distinguished itself with a growth rate that allows colonies to grow to adequate size for visual confirmation within the normal incubation time.

Figure 3. Comparison of colony growth rate on agar after transformation. DNA plasmids of 2 kb (Panel A) and 10 kb (Panel B) were each used to transform Stellar and DH10B competent cells with similar genetic characteristics, including methylation requiring restriction. This experiment used the same method as in Figure 1. Photographs of colonies on agar were taken after 15 hours of incubation.

Transformation efficiencies are provided by vendors as a gauge of competent cell performance. In real-life experiments however, actual transformation efficacies may be substantially lower than those listed. Moreover, ligation reaction mixtures are generally used as-is in transformations, with buffer compositions introducing additional problems. Certain ingredients may inhibit transformation and further reduce efficiency to an inadequate level. It is thus very difficult to make an appropriate determination regarding the performance of a competent cell strain based entirely on the transformation efficiency listed in the catalog.

In these experimental examples, Stellar Chemically Competent Cells demonstrated high efficiencies in transformations with both purified plasmids and ligation reaction mixtures. Stellar Competent Cells are therefore an excellent choice for a variety of cloning projects.