You've worked hard designing your plasmid – you carefully selected the optimal promoter for your gene of interest, you painstakingly cloned into the perfect empty backbone, you made sure to add the right tags and an NLS to your gene, you put a fluorescent protein downstream, separated by an IRES element. You did a lot of work! But let’s take a moment to recognize your little prokaryotic minions that carried out the labor-intensive process of replicating your new plasmid: the Escherichia coli bacterium.
It’s hard to count the number of different commercial strains of E. coli currently available – a quick Google search suggests there are hundreds. This only includes general lab strains designed for subcloning or protein expression. If you were to include customized strains, the number is probably in the thousands! The goal of this article is to provide enough background for you to distinguish the features of any common lab strain and determine whether it is appropriate for propogating your plasmid or carrying out your experiment.
History of E. coli Strains
E. coli are gram-negative, rod shaped bacteria that were named after Dr. Theodor Escherich, the scientist who first described them in 1885. E. coli are mainly found in the intestinal tract of animals. There are many different naturally occurring strains of E. coli, some of which are deadly to humans. The majority of all common, commercial lab strains of E. coli used today are descended from two individual isolates, the K-12 strain and the B strain. K-12 was isolated from a patient in 1920 and eventually led to the common lab strains MG1655, which led to DH5alpha and DH10b (also known as TOP10). The history of B strain is a bit more convoluted due to researchers sharing and renaming samples throughout history. It was likely isolated in 1918 but was first referred to as “B strain” in 1942. The BL21 strain, (and derivatives) are the most common examples of the E. coli B strain.
Common E. coli Strains Used in the Lab
Most of the commercial strains you find today are marketed for a specific purpose: fast growth, high-throughput cloning, routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that make these features possible are present in most commercial strains, especially mutations that make major improvements such as those that increase plasmid yield and/or DNA quality. Table 1 below outlines a few of the more common genetic changes found in E. coli strains.
TABLE 1 - Common gene mutations found in E.coli strains
|dam||DNA Adenine methylase mutation (GATC)||Preparing unmethylated DNA, important when trying to cut with certain restriction enzymes (ex: ClaI or XbaI)|
|dcm||DNA Cytosine methylase mutation (CCWGG)||Preparing unmethylated DNA, important when trying to cut with certain restriction enzymes that are methylation sensitive.|
|dnaJ||Mutation in a chaperonin gene||Increases the stability of certain expressed proteins|
|endA, endA1||Endonuclease I (nonspecific cleavage of dsDNA ) mutation||Improves plasmid yield|
|F||Host does (F') or does not (F-) contain the fertility plasmid.||A low copy-number plasmid, encodes proteins needed for bacterial conjugation. Genes listed on F´ are wild-type unless indicated otherwise|
|fhuA (formerly tonA)||ferric hydroxamate uptake, iron uptake receptor mutation.||T1/T5 Phage resistance|
|gal||Mutation in galactose metabolism pathway||cells cannot grow on just galactose|
|gyrA, gyrA96||DNA gyrase mutation||Confers resistance to nalidixic acid|
|hsdRMS||hsdR(rk-, mk+)||unmethylated DNA not degraded, cell still can methylate DNA|
|hsdS(rk-,mk-)||unmethylated DNA not degraded, cell cannot methylate DNA|
|lac||Lac Operon Mutations||blue/white screening of clones|
|lacIq||lac repressor overproduced, expression from pLac repressed more|
|LacZ||β-galactosidase activity abolished|
|lacY||Lactose permease inactivated, lactose cannot be taken up by cell|
|mcrA, mcrBC||Inactivation of pathway that cleaves methylated cytosine DNA||Allows for uptake of foreign (methlyated) DNA|
|mrr, Δ(mcrC-mrr)||Inactivation of pathway that cleaves methylated adenine or cytosine DNA||Allows for uptake of foreign (methlyated) DNA|
|recA, recA1, recA13||Mutation in a DNA-dependent ATPase that is essential for recombination and general DNA repair||Reduces plasmid recombination, increases plasmid stability|
|recBCD||Exonuclease V activity abolished||Increased plasmid stability, reduced recombination|
|relA or relA1||Relaxed phenotype, mutation eliminating stringent factor||Allows RNA synthesis in absence of protein synthesis|
|Ptrc-ccdA||Propagation of ccdB-containing plasmids|
|Hte||"high transformation efficieny"|
|deoR||constitutive expression of genes for deoxyribose synthesis||Allows uptake of large plasmids|
|hee||"high electroporation efficiency"|
|supE44 (glnV44)||Suppression of the amber (UAG) stop codon by inserting glutamine|
|supF (tyrT)||Suppression of the amber (UAG) stop codon by inserting tyrosine|
|λ-thi-1 or thi1||Mutation in thiamine metabolism||requires exogenous thiamine for growth|
|ara||disruption of arabinose metabolism pathway||inability to utilize arabinose as a carbon source|
|leuB||β-isopropyl malate dehydrogenase inactivated||requires exogenous leucine source for growth|
|proAB||mutation in proline biosynthesis pathway||requires exogenous proline source for growth|
|rpsL||Mutation in subunit S12 of 30S ribosome||Confers resistance to streptomycin|
|Tn10||Confers resistance to tetracycline|
Additionally, Table 2 provides a quick reference of some of the popular strains, their genotypes, and their primary use in the lab. These strains are all based on E. coli K-12 and are considered the lowest biosafety level.
TABLE 2- Lab strains of E. coli
|Strain||Natural resistance||Primary Use||Genotype|
|ccdB Survival||For propagating plasmids expressing the ccdB gene (important in Gateway cloning).||F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG tonA::Ptrc-ccdA|
|DB3.1||Streptomycin||For propagating plasmids expressing the ccdB gene (important in Gateway cloning).||F- gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(rB-, mB-) ara14 galK2 lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1
|DH10B||Streptomycin||MC1061 derivative. General cloning and storage, blue/white screening, leucine auxotroph.||F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ-|
|DH5alpha||General cloning and storage of common plasmids, blue/white screening.||F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–|
|HB101||Streptomycin||Hybrid of E. coli K12 and E. coli B (mostly K12, though), common lab strain for cloning and storage of pBR322 plasmids.||F- mcrB mrr hsdS20(rB- mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ-|
|JM109||General cloning and plasmid maintainence, blue/white screening, partly restriction-deficient; good strain for cloning repetitive DNA.||endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36 proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)|
|JM110||Streptomycin||For storing plasmids that should not be methylated, allows for methylation sensitive restriction enzymes to cut the plasmid after preparation.||rpsL thr leu thi lacY galK galT ara tonA tsx dam dcm glnV44 Δ(lac-proAB) e14- [F' traD36 proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)|
|MC1061||Streptomycin||Parent of DH10B/TOP10 and derived strains, common lab cloning and storage strain.||F- Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 λ- e14- mcrA0 relA1 rpsL150(strR) spoT1 mcrB1 hsdR2(r-m+)|
|MG1655||"wild type" K-12 strain.||F- λ- ilvG- rfb-50 rph-1|
|Pir1||For cloning and maintenance of a plasmids with R6Kγ origin; contains a mutant allele of the pir gene that maintains the plasmids at ~250 copies per cell.||F- ∆lac169 rpoS(Am) robA1 creC510 hsdR514 endA recA1 uidA(∆MluI)::pir-116|
|Stbl2||JM109-derived. For storage of plasmids that have the potential to recombine. Example, the LTRs in lenti- and retro-viral plasmids.||F- endA1 glnV44 thi-1 recA1 gyrA96 relA1 Δ(lac-proAB) mcrA Δ(mcrBC-hsdRMS-mrr) λ-|
|Stbl3||Streptomycin||Derived from HB101. For storage of plasmids that have the potential to recombine. Example, the LTRs in lenti- and retro-viral plasmids, endA+, use care in preparing DNA from this strain.||F- glnV44 recA13 mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1|
|Top10||Streptomycin||MC1061 derivative. General cloning and storage, blue/white screening.||F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-|
|XL1 Blue||Tetracycline||Blue/white screening and routine cloning, nalidixic acid resistant.||endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+)|
|XL10 Gold||Tetracycline and Chloramphenicol||Cloning and propagation of large plasmids, high competency, nalidixic acid resistant.||endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 tetR F'[proAB lacIqZΔM15 Tn10(TetR Amy CmR)]|
Note: generally inactivating mutations in specific genes are signified with a minus sign (-) as is typically standard; just having the gene listed indicates it is non-functional. If a gene is deleted that is normally noted with a Greek delta (Δ).
We've provided an overview of the common lab strains; however, these tables are by no means exhaustive! For a more comprehensive list and additional information, visit OpenWetWare's E.coli genotype resource. Additionally, NEB has a great list of genetic markers for your reference.
Browse Addgene's curated list of Bacterial Expression Systems.
And check back soon for our companion post describing protein expression strains, where we’ll go over the basics of protein expression in E. coli and the common features found in those bacteria.
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