Competent Cell Selection Guide

Find more protocols and selection guides in the Molecular Biology Guide.


Transformation is a process by which some bacteria take up foreign genetic material (naked DNA) from the environment. Once it enters the cytoplasm, the genetic material may be degraded by nucleases if it is different from the bacterial DNA. If the exogenous genetic material is similar to bacterial DNA, it may be integrated into the chromosome. Sometimes the exogenous genetic material may co-exist as a plasmid with chromosomal DNA.

Not all bacteria are capable of taking up exogenous DNA from their environment.  For the bacterium to uptake DNA, it has to be competent. Bacteria can be naturally competent or made competent by artificial methods. The factors that regulate natural competence vary between various genera. The practical approach to acquire competent cells is to make the bacterial cells artificially competent using chemicals or electrical pulses.

  • Induction of competence chemically involves the following steps:
    • Chilling the cells in the presence of calcium chloride or magnesium chloride to make them permeable
    • Incubation with DNA
    • Heat shock treatment at 42° C for 60-120 seconds that causes the DNA to enter the cells

It is important that the cells used are in the log phase of growth to endure the heat shock treatment.

  • Alternatively, the bacterial cells are made permeable by subjecting them to electrical pulses, a process known as electroporation.

The phenomenon of transformation has been widely used in molecular biology. Bacteria may be used as host cells to make copies of the DNA, cloning, to express large amounts of proteins, generation of cDNA libraries and in DNA linkage studies as they are easily grown in large numbers.

Sigma Competent Cell Selection Guide

Sigma-Aldrich offers a range of Escherichia coli bacterial cells made competent by optimized procedure specific to each strain, involving strain verification and efficiency testing. Our current competent cells are being discontinued; soon we will offer a wider variety of competent cells with higher transformation efficiency.

Key to Genotypes

Genotype Description
hsdR17 Mutation that inhibits microbe sensing presence of foreign DNA
F’ episome Self-transmissible, low copy number plasmid used to generate ssDNA when infrected with M13 bacteriophage
D(lacZ)M15 Allows for blue/white screening
ton A Resistance to bacteriophages T1, T5 and f80
ompT- Lacking outer membrane protease, which reduces degredation and improves protein recovery
pLysS Encodes the T7 lysozyme; used to reduce basal expression by inhibiting RNA polymerase
CmR Resistance to chloramphenicol
endA1- Mutation in Endonuclease I, eliminating non-specific activity, improving plasmid preps
T1R Resistance to T1 bacteriophage
recA1-
DNA recombination gene mutation – useful for cloning genes with direct repeats

 

 References

  • Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular cloning: a laboratory manual. New York: Cold spring harbor laboratory press, 1989.
  • Dubnau, D., DNA uptake in bacteria. Annu Rev Microbiol., 53:217-44 (1999).
  • Lorenz, M.G., and Wackernagel. W., Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev., 58(3):563-602 (1994).