Genotypes and Genetic Markers of Competent E. coli Cells

• Amber (UAG) mutation
• See also supE and supF

• Resistant to ampicillin

• ara-14strain carries mutation resulting in inability to metabolize arabinose
• araD: This gene encodes ribulose-5-phosphate 4-epimerase, which is one of the enzymes needed for metabolism of the sugar L-arabinose in E. coli. Strains with an araD mutation cannot metabolize L-arabinose.

• Chromosomal deletion of (b0059) b0060-b0079 genes. The strain lacks the leuLABCD operon (this operon encodes the enzymes responsible for biosynthesis of leucine from valine), for example, DH10B strain bearing this mutation is unable to grow on synthetic minimal medium (requires amino acid supplements).

• Mutation in the ornithine carbamoyltransferase gene
• Blocks ability to make arginine

• Parent bacmid (baculovirus shuttle vector) in DH10Bac
• A low-copy number mini-F replicon
• Kanamycin resistance marker
• A segment of DNA encoding the LacZα peptide from a pUC-based cloning vector into which the attachment site for the bacterial transposon, Tn7 (miniattTn7) has been inserted. Insertion of the mini-attTn7 does not disrupt the reading frame of the LacZα peptide.

• Resistant to chloramphenicol

• Encodes two genes from the F′ episome for control of cell death
• Allows positive selection of colonies from successful cloning
• (The ccdB protein causes gyrase-mediated DNA cleavage, while ccdA negates ccdB toxicity.)

• Mutation of a gene involved in phosphate limitation
• Expresses CreC constitutively

• Mutation in DNA adenine methyltransferase (an enzyme that methylates adenine in GATC sequence, in double stranded DNA), resulting in lack of adenine methylation, high recombination efficiency and activation of DNA repair.
• Allows restriction with methylation sensitive enzymes

• Mutation in DNA cytosine methyltransferase which methylates the second C of CCWGG, preventing cleavage of DNA by restriction enzymes. Mutation is indicated with dcm-.
• Allows restriction with methylation sensitive enzymes

• Strain carries the DE3 phage encoding for T7 RNA polymerase. Useful when inducing protein expression using T7 promoter expression systems.

• Mutation results in a lack of Endonuclease I (nonspecific cleavage of dsDNA)
• Allows for foreign plasmid cloning as intracellular endonucleases are absent, thus preventing its degradation
• Improves quality of isolated plasmid DNA

• Strain does not contain an F episome (also called fertility factor)

• F⁺ indicates that the bacterium carries the F plasmid (also called fertility factor). However, the F plasmid is not inserted into the bacterial genome.
• F’[ ] indicates the F plasmid has inserted into the bacterial genome, then has gone through excision, becoming an independent plasmid. During excision, some of the bacterial genome sequences were excised with the F plasmid. Thus, this indicates that the F plasmid exists outside of the bacterial genome and carries bacterial genes, which are specified inside the brackets.
• Hfr denotes high frequency of recombination. Indicates that the F plasmid has inserted (integrated) into the bacterial genome.
• F episome encodes strand-like structure called pili on the outer membrane of E. coli
• May carry lacI^q, lacZ∆M15, and an antibiotic resistance marker
• Allows M13 phage infection
• Enables ssDNA generation

• Mutation in the ferric hydroxamate uptake (also known as ferrichrome outer membrane transporter) gene

• Mutation in galactose metabolism pathway
• Strain cannot grow using galactose as the sole carbon source

• Gene product is galactokinase, which catalyzes the phosphorylation of galactose to galactose-1-phosphate. Mutation results in lack of galactose metabolism.
• Strain cannot grow using galactose as the sole carbon source

• Mutation in glucose-1-phosphate uridylyltransferase leading to lack of galactose metabolism
• Strain cannot grow using galactose as the sole carbon source

• Mutation in DNA gyrase (an enzyme that allows ATP-dependent negative supercoiling of double-stranded circular DNA)
• Mutation results in nalidixic acid (a bactericidal that inhibits replication in bacteria) resistance

• Mutations in the methylation and restriction system genes
• Allows cloning of DNA from non-E. coli sources, such as PCR products, without cleavage by endogenous restriction endonucleases
• The genetic marker may contain the allele number (e.g., hsdR17) and phenotype (e.g., (r_K^–, m_K⁺), where r is for restriction, m for methylation, and the subscript _K for the parental strain E. coli K12)

• Insertion element 2

• Resistant to kanamycin

• Lambda phage infects bacteria and is routinely used as a vector in cloning
• λ^— denotes lambda phage deletion

• Mutation (-35 site in promoter of lacI, GCGCAA to GTGCAA) results in constitutive expression of lac repressor and inhibition of the lac promoter
• Allows controlled gene expression from promoters that carry the lac operator
• Inhibits transcription from the lac promoter, which can be overcome by IPTG addition
• The superscript ^q indicates a constitutive mutation

• Lactose permease mutation resulting in deficient lactose transport
• Blocks lactose utilization

• Deletion of the lac operon

• Deletion of a chromosomal region including the lac operon and argF in the allele U169
• Deletion exhibits a high level of resistance to hydrogen peroxide compared with its undeleted parent

• Partial deletion of lacZ (β-galactosidase) gene that allows α-complementation
• Enables blue-white screening for recombinant colonies when plated on X-Gal/IPTG

• Strain requires leucine for growth on minimal medium

• Mutation in the lon (ATPase-dependent protease) gene
• Lon is a cytoplasmic protease that degrades aberrant proteins in bacteria in response to stress
• ∆lon results in lack of proteolysis and may facilitate expression of a protein of interest. However, Lon may be useful in membrane protein overexpression.

• Mutations in the methylation-dependent restriction system (MDRS) genes
• Indicates DNA methylation. McrA and McrBC recognize methylcytosine whereas Mrr recognizes both methylcytosine and methyladenine. Mutation results in inactivation of cleaving of DNA with methylated cytosine (mutation of mcrA, mcrBC, mrr) or adenine (mutation of mrr) and allows cloning of methylated DNA.
• Allows for efficient cloning of methylated DNA from eukaryotic sources

• Enables cadmium ion binding activity and zinc ion binding activity

• Resistant to nalidixic acid

• Mutation in a nucleoside transport gene
• Increases plasmid uptake

• Mutation in the ompT (outer membrane protease) gene
• Reduces degradation of expressed recombinant proteins

• Carries lambdoid prophage φ80

• 60 kb low-copy plasmid
• Harbors ampicillin and tetracycline resistance genes with amber mutations
• Allows selection of supF-containing plasmids
• Confers kanamycin resistance

panD

• In strains expressing the panDE.c. gene, the maximal pantothenate production is dependent on external beta-alanine supplementation

• Mutation in the alkaline phosphatase gene
• Blocks phosphate utilization

• The pir gene encodes the replication protein π, which is required to replicate and maintain plasmids containing the R6Kγ origin
• Wild-type pir gene that maintains the donor at ~15 copies per cell
• pir-116 carrying strain maintains the donor vector construct at ~250 copies per cell

• Plasmid that encodes T7 lysozyme
• Inhibits binding of T7 RNA polymerase to the T7 promoter
• Reduces basal expression of cloned genes driven by the T7 expression system
• Confers chloramphenicol resistance

• pBR322-derived helper plasmid in DH10Bac
• Carries transposase genes for insertion of the donor plasmid mini-Tn7 into the parent bacmid bMON14272

• Mutation in proline metabolism genes A and B
• Strain requires proline for growth on minimal medium

• Mutations in genes for general recombination of DNA
• Wild-type RecA protein mediates DNA repair and homologous recombination.
• Enables cloning of unstable DNA (e.g., sequences with repeats)

• Relaxed phenotype, mutation eliminating stringent factor

• Encodes T7 RNA polymerase

• Mutation in the RNase E gene
• Helps prevent mRNA degradation
• Stimulates expression from a limited number of genes

• Mutation in the right oriC-binding protein gene

• Mutation in the RNA polymerase sigma factor gene
• Abolishes expression of some stress-induced proteases
• Improves yield of certain recombinant proteins at high temperature

• Mutation in the 30S ribosomal protein small subunit S12 gene
• Confers resistance to streptomycin (which targets the 30S subunit)

• Resistant to streptomycin

• Suppresses the amber (UAG) mutation
• Suppressor tRNA inserts glutamine at the mutation site
• Required for growth of strains with amber mutations and phage display systems

• Resistant to tetracycline

• Mutation in a thiamine metabolism gene
• Strain requires thiamine for growth on minimal medium
• The hyphen in thi-1 indicates the exact locus is unknown

• Mutation in a threonine metabolism gene
• Strain requires threonine for growth on minimal medium

• Transposon that confers resistance to kanamycin

• Transposon that confers resistance to tetracycline

• Mutation in an outer membrane iron uptake receptor gene
• Confers resistance to the lytic bacteriophage T1, T5, and φ80

• Mutation in a transfer factor gene
• Prevents transfer of F′ episome

• Confers resistance to phage T6 and the polypeptide bacteriocin colicin K

• Mutation in the β-glucuronidase gene between MluI restriction sites

• Mutation in a xylose metabolism gene
• Blocks xylose utilization

• Insertion (::) of Tn5 on the F plasmid
• zzf denotes an insertion mutation on the F′ episome