Cloning & Expression

Guide to E. coli Markers

In the descriptions below, the markers are described as the genes or gene products.

dam  Methylates A of GATC sites. The dam methyl pattern is used to distinguish old (methylated) from new (unmethylated) strands for damage-repair purposes (repair the unmethylated strand). Dam methylation is also used to time replication (don’t replicate until both strands are dam methylated). This methylation interferes with several restriction enzymes, including BclI and ClaI.

dcm  Methylates CCWGG.

dnaJ  Chaperonin. Mutants can stabilize some proteins in E. coli.

dut  dUTPase, an enzyme that prevents the incorporation of uracil into DNA by destroying dUTP. Dut-ung double mutants accumulate a significant amount of uracil in their DNA.

e14  A prophage-like element, present in K-12 but missing from many derivatives. e14 carries the mcrA gene, so e14- strains are McrA-.

endA  The gene for the end1 nuclease, the primary endonuclease of E coli.

F  A self-transmissible plasmid (100 kb) that confers the ability to make pili (i.e. be "male") and thus to be infected by male-specific phage like M13.

F'  An F plasmid that picked up some chromosomal DNA from E. coli. The F’ can have its own genotype. Two popular models code for the lacβ peptide (ΔM15), or LacIQ.

hsdRSM  Restriction system that methylates host DNA in specific sequences and cleaves DNA that is not as methylated. The R gene is the endonuclease, the M gene is the methylase, and the S gene is required for each to function. Thus hsdR mutants don’t have the endonuclease function, but still methylate. HsdS mutants do not have the endonuclease or the methylase. Two popular models are the K system of E. coli K, and the B system of E. coli B.

lacIQ  Overproduces the lacI gene product, a repressor of the lac operon.

lacY  Lactose permease. LacY mutants cannot be used in blue/white screening, but are used to regulate gene expression in IPTG-induced gene expression.

lacZ  β-galactosidase gene. The Δ(lacZ)M15 mutation expresses the β fragment. This is commonly found on F’ elements or in a defective 80 φ prophage. Many plasmids express the α fragment. The two together form a functional β-galactosidase.

Δlac  There are three common deletions involving the entire lacZYA operon in addition to some flanking DNA: ΔU169, Δ X111, and ΔX74.

Ion  A protease responsible for degrading aberrant proteins and for turning off sulA function. E. coli B naturally lacks lon.

mal B  The malB region encompasses the genes malEFG and malK lamB malM. Δ (malB) deletes most or all of this entire region. LamB is the receptor for lambda phage.

mcr A  Restricts DNA with methylcyctosine in certain sequences. McrA is lost when prophage e14 is lost.

mcr BC  A system that restricts DNA containing methylcytosine in certain sequences. Δ(mcrC-mrr) deletes six genes: mcrC-mcrB-hsdS-hsdM-hsdR-mrr

mrr  A methylcytosine and methyladenine restriction system.

recA  Required for most homologous recombination pathways.

recB  Required for ExoV function. Recombination deficient.

recC  Required for ExoV function.

recD  Required for ExoV function. RecD mutants stabilize inverted repeats, but interfere with plasmid maintenance.

recF  Recombination gene required for interplasmid homologous recombination.

recJ  Recombination gene required for interplasmid homologous recombination.

rpoH  (or htpR heat shock transcription factor) Required to express some heat shock proteases.

sbcB  Required for Exonuclease I function. Strains carrying recB recC and sbcB are usually also sbcC. These quadruple mutant strains are recombination-proficient and propagate inverted repeats in λ, but plasmid replication is aberrant.

sbcC  Helps (with sbcB) to supress the effect of a recBC mutant. sbcC mutants are Rec+ and stably propagate inverted repeats in plasmids.

supE  (or glnV) Mutant tRNA inserts glutamine at UAG codons, suppressing UAG mutations in the reading frame. SupE is required for the lytic growth of some phage mutants.

supF  (or tyrT) Mutant tRNA inserts tyrosine at UAG codons, thus suppressing the effect of UAG mutations in reading frames. This mutation is required for the lytic growth of some λ phage, such as λgt11.

traD  Conjugation defective mutant of the F plasmid.

ung  Mutant in uracil N-glycosylase, an enzyme that removes uracil from DNA. An ung mutant allows uracil to persist in DNA.

φ80dlacΔM15  Lysogenic for a defective prophage derivative of φ80. The lacZ ΔM15 allele supplies the beta peptide for blue white screening.

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