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Choosing a Competent E.coli Strain

Posted in: DNA / RNA Manipulation and Analysis

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Choosing a Competent E.coli Strain

Of all the competent E. coli cell strains available (including both chemically competent or electrocompetent E. coli), which one should you choose? The choice of strain to use in a given experiment is determined in large part by the nature of the experiment and the set of traits that best fit it. In this article I summarize some of the most important traits and their benefits in downstream applications.

Note that when writing out a strain’s genotype, one usually lists only the known mutations and everything else is assumed wild-type. The mutant alleles are not given a minus sign, e.g. endA describes the endA null phenotype. Deletions are indicated by a delta before the mutant allele.

This is the third of three articles on E. coli competent cells and transformation. Part 1, Part 2

Traits that maintain the integrity of the transformed plasmid
endAKnock-out mutation in non-specific endonuclease (Endonuclease I). Eliminates non-specific endonuclease activity.Improved plasmid yield/quality
hsdRMutations in hsdR prevents restriction of unmethylated EcoKI sitesEfficient transformation of DNA generated from PCR reactions
dam/dcmMutations in dam/dam abolish adenine and cytosine methylation at specific recognition sequences.Propagation of DNA for cleavage with methylation-sensitive restriction enzymes e.g. Ava II, Bcl I
mcrA, mcrBC,or mrrMutations in these genes prevents methylated DNA from other organisms from being recognized as foreignAllows cloning of genomic DNA or methylated cDNA
recAMutation in recA reduces DNA recombinationIncreased plasmid DNA stability
recBCDrecBCD encodes exonuclease V. Mutation in RecB or RecC reduces DNA recombination by a factor of 100.Increased plasmid DNA stability
recJ/sbcC Also involved in DNA recombination. Inactivation increases plasmid DNA stabilityIncreased plasmid DNA stability
uvcR/umuCInvolved in the UV and SOS DNA repair systems respectively. The inactivation of these genes increases the stability of plasmids carrying inverted repeatsIncreased plasmid DNA stability
Traits for the identification of positive clones
lacZ-delta-M15Deletion of the N-terminal alpha-fragment from the LacZ gene, making ?-galactosidase function dependent on expresion for the lacZ-? fragment from another source (e.g. a plasmid)Used for blue/white screening of recombinant plasmids carrying the lacZ-? fragment
lacIThe lac repressor. Inhibits expression from the Lac promoter in the absence of lactose/IPTGA functional lacI is required for blue/white screening
Traits for improved transformation efficiency
deoRDeletion of a regulatory gene, allowing constitutive expression of deoxyribose synthesis geneIncreases the transformation efficiency for large plasmids
heeStands for “high electroporation efficiency”. Increases survival rate of cells during electroporation, leading to higher transformation efficiency.High transformation efficiency
hteStands for “high transformation efficiency”.High transformation efficiency.
Traits Related to Protein Expression
lacIqThis mutated version of LacI gives high levels of lac repressor expression. This allows tighter regulation of gene expression from the lac promoterTightly regulated gene expression from lac promoter
DE3Lysogen that encodes T7 RNA polymerase. Used to induce expression in T7-driven expression systemsRequired for expression from the T7 promoter in E.coli
pLysSpLysS is normally plasmid borne. It harbors T7 lysozyme, which destroys T7 polymerase produced from DE3. Used to reduce basal expression in T7-driven expression systems by inhibiting basal levels of T7 RNA polymeraseTightly regulated gene expression from T7 promoter
lonDefficiency in the Lon ATPase-dependent protease. Decreases the degradation of recombinant proteins; all B strains carry this mutationReduced degradation of recombinant proteins
ompTDefficiency in an outer membrane protease.Reduced degradation of recombinant proteins
araD/ara-14Cannot metabolize arabinoseEnhances expression from araB promoter
dnaJ encoded chaparonin is inactivatedImproves folding of some heterologous proteins
Mutation in glutathione reductase, which enhances disulphide bond formationImproves folding of heterologous proteins requiring disulphide bonds

The table below has a few suggestions of competent cell strains to use for some applications. All of these strains are available from Invitrogen or Stratagene, although many other manufacturers make the same or equivalent strains:

Routine Cloning/Sub-cloning, Blue/white screeningXLI-Blue, DH5-alpha, top10
Very high efficiency cloning e.g. for library constructionXL10-Gold, MegaX DH10b
Cloning of unmethylated DNAXL1-Blue MR
Production of unmethylated DNAJM110, INV110
Cloning of unstable plasmidsSure, Stbl4
Expression from T7 promoterBL21 (DE3)
Expression from T7 promoter, tight regulationBL21 (DE3) pLysS
Expression from T7 promoter with codon bias correctionBL21 codon plus, Rosetta
Improved disulphide bond formationOrigami
Fast cloning (due to quick cell growth)Mach1

For a constant supply of high-quality competent cells, download bitesize bio’s chemically competent cells protocol cheat sheet—your reliable set of instructions to prepare chemically competent cells in the lab.

Articles in this series:

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