Choosing a Competent E.coli Strain |
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Nick Oswald |
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Of all the of competent E. coli cell strains available, 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 |
dnaJ encoded chaparonin is inactivated | Improves folding of some heterologous proteins |
| gor |
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 |
Articles in this series:
Photo: Shades of Mediocrity
E.coli Electroporation vs Chemical Transformation
Competent E.coli: To buy or not to buy?
DIY Electrocompetent E. coli
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EmWed
Fabulous table- very useful!
micheal
the way of your lab of chemical transformation is suitable for xl10-gold?
Nick
It is possible to prepare home-made chemically competent XL-10 gold using the normal procedures. I hope this answers your question.
Rhincodon
Hi,
I recommend SHuffle cells from NEB, for the expression of disulfide-rich proteins in E.coli. Uses less antibiotics than the Origami strain, and growth rate is like good old BL21.
Melissa
Hi Nick,
Do you happen to know if this protocol for making electrocompetent cells can be applied to Acinetobacter baumannii?