Choosing the right competent cell line for your assay and achieving a successful transformation are dependent on understanding genotypes and genetic markers. Here, we describe the nomenclature of E. coli genotypes to help guide you in choosing your competent cells.
In This Article:
Genes and genotype in E. coli :
Restriction-modification system:
What is a genotype
The genotype (genes) of an organism, including E. coli, is expressed resulting in a phenotype, which is an observable and measureable characteristic. The genotype will contain information for genes, mutation types, allele numbers, antibiotic resistance, insertions, fusions, inversions, plasmids and deletions carried by a specific bacterial strain.
Genes and genotype in E. coli :
In E. coli, the genotype only includes the genes that carry a loss of function mutation.
The gene name is listed as three-letters in lower case and italics (DNA methylase is written as dam). Different genes affecting the same function/pathway are identified with different uppercase italic letters. GalE and galK are examples of genes encoding proteins that function in the same pathway.
In the case of different mutations in the same gene, or
different alleles (genes can have variants, which are known as alleles), a
number is added to indicate the specific mutation (allele).
The presence of a (q) indicates a constitutive mutation.
Insertions:
Transposable elements have their own mechanism to move and insert themselves into the host organism's DNA. Insertion of a transposon into a known gene is indicated as gene::transposable element.
Deletions:
A deletion of a specific gene is indicated with ∆ and stated as ∆gene, where ∆ is placed before the gene name. If a deletion encompasses multiple genes, then it is written as ∆(gene-gene) indicating that a whole region including the genes listed and those in that segment are deleted.
Fusions:
The fusion of different elements is often indicated by ɸ(fused element-fused element), by a colon (:) or simply by a dash (-).
Inversion:
A chromosomal inversion of a segment between two specific sites is indicated by INV(gene-gene). See Figure 1.
Figure 1. Inversion of a chromosomal segment containing genes B, C and D.
Plasmids:
The inclusion of specific plasmids can be indicated in the genotype. The F plasmid is a DNA plasmid called Fertility Factor or Sex Factor. This plasmid carries the genes that allows the transfer of plasmid DNA from one cell to another. Confers the ability to mate with F- through conjugation.
Prophages:
A prophage, also known as a “phage,” can infect bacterial cells resulting in its genome integrating into the bacterial DNA chromosome (lysogenization) or becoming an extrachromosomal plasmid (lytic growth).
Restriction-modification system:
E. coli have restriction (cleavage of DNA) and modification (by methylation) systems that confer protection from foreign DNA.
The hsdR, hsdM and hsdS genes encode an EcoKI restriction-modification system which degrades DNA sequences that are not methylated at the adenine at 5’-AACN6GTGC-3’. These sequences are degraded by the EcoKI restriction enzyme.
On the other hand, McrA, McrBC and Mrr are part of different restriction-modification systems in which restriction (digestion) occurs if DNA methylation is present. 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.
Other Phenotypic Traits
Relevant phenotypic characteristics, including antibiotic resistance and sensitivity to cold, can also be described in the genotype using the following specific nomenclature.
Antibiotic resistance:
Resistance to a specific antibiotic is indicated by two, three or four letters for the antibiotic and a superscript uppercase R (R).
Sensitivity:
Conditional alleles with different sensitivities are indicated in the genotype. Sensitivity to cold and temperature are indicated as lowercase superscript cs (cs) and ts (ts), respectively. Temperature sensitivity indicates that exposure to high temperature results in inactivation. Cold sensitivity results in a loss-of-function phenotype only at low temperatures (lower than a specific threshold temperature).
Other conditional alleles:
Other mutations can be indicated with two lowercase superscript letters. Examples are the amber mutation, the opal mutation and the ochre mutation, which introduce a stop codon and results in a truncated protein product.
Relevant Genetic Markers
References
Casali, N., & Preston, A. (2003). E. coli Plasmid Vectors: Methods and Applications. Totowa, NJ: Humana Press Inc.
E. coli Genetic Resources at Yale CGSC, The Coli Genetic Stock Center. (n.d.). Retrieved November 13, 2019, from https://cgsc.biology.yale.edu/.
E. coli genotypes. (n.d.). Retrieved November 13, 2019, from https://openwetware.org/wiki/E._coli_genotypes#References.
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