Plasmids 101: Common Lab E. coli Strains

Posted by Matthew Ferenc on Nov 7, 2014 9:56:00 AM


minion

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

Bacterial_Plate

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

Gene(s) Description Functional Consequence
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). 

Additional Resources

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.


Our Most Popular Plasmids 101 Posts:

Looking for More Plasmids 101 Articles? 

Find them all here: http://blog.addgene.org/topic/plasmids-101


  Click to Download The Plasmids 101 eBook (2nd Edition) Don't miss the next Plasmids 101 post! Click here to subscribe to Addgene's Blog.

 

Topics: Lab Tips, Plasmids 101

Addgene blog logo

Subscribe to Our Blog