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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
Genotype Description Benefit
endA Knock-out mutation in non-specific endonuclease (Endonuclease I). Eliminates non-specific endonuclease activity. Improved plasmid yield/quality
hsdR Mutations in hsdR prevents restriction of unmethylated EcoKI sites Efficient transformation of DNA generated from PCR reactions
dam/dcm Mutations 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 mrr Mutations in these genes prevents methylated DNA from other organisms from being recognized as foreign Allows cloning of genomic DNA or methylated cDNA
recA Mutation in recA reduces DNA recombination Increased plasmid DNA stability
recBCD recBCD 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 stability Increased plasmid DNA stability
uvcR/umuC Involved in the UV and SOS DNA repair systems respectively. The inactivation of these genes increases the stability of plasmids carrying inverted repeats Increased plasmid DNA stability
Traits for the identification of positive clones
Genotype Description Benefit
lacZ-delta-M15 Deletion 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
lacI The lac repressor. Inhibits expression from the Lac promoter in the absence of lactose/IPTG A functional lacI is required for blue/white screening
Traits for improved transformation efficiency
Genotype Description Benefit
deoR Deletion of a regulatory gene, allowing constitutive expression of deoxyribose synthesis gene Increases the transformation efficiency for large plasmids
hee Stands for “high electroporation efficiency”. Increases survival rate of cells during electroporation, leading to higher transformation efficiency. High transformation efficiency
hte Stands for “high transformation efficiency”. High transformation efficiency.
Traits Related to Protein Expression
Genotype Description Benefit
lacIq This mutated version of LacI gives high levels of lac repressor expression. This allows tighter regulation of gene expression from the lac promoter Tightly regulated gene expression from lac promoter
DE3 Lysogen that encodes T7 RNA polymerase. Used to induce expression in T7-driven expression systems Required for expression from the T7 promoter in E.coli
pLysS pLysS 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 polymerase Tightly regulated gene expression from T7 promoter
lon Defficiency in the Lon ATPase-dependent protease. Decreases the degradation of recombinant proteins; all B strains carry this mutation Reduced degradation of recombinant proteins
ompT Defficiency in an outer membrane protease. Reduced degradation of recombinant proteins
araD/ara-14 Cannot metabolize arabinose Enhances expression from araB promoter
dnaJ encoded chaparonin is inactivated Improves folding of some heterologous proteins
Mutation in glutathione reductase, which enhances disulphide bond formation Improves 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:

Application Strain
Routine Cloning/Sub-cloning, Blue/white screening XLI-Blue, DH5-alpha, top10
Very high efficiency cloning e.g. for library construction XL10-Gold, MegaX DH10b
Cloning of unmethylated DNA XL1-Blue MR
Production of unmethylated DNA JM110, INV110
Cloning of unstable plasmids Sure, Stbl4
Expression from T7 promoter BL21 (DE3)
Expression from T7 promoter, tight regulation BL21 (DE3) pLysS
Expression from T7 promoter with codon bias correction BL21 codon plus, Rosetta
Improved disulphide bond formation Origami
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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