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Selection refers to applying conditions that allow the desired cells or phages (containing vector or vector and insert) to replicate while preventing others from replicating. It is one of five requirements for efficient molecular cloning in E. coli.

Facts

• E. coli bacteria wer grown in nutrient broth to about 10⁸ cells/ml. 0.1 ml was spread on a plate of nutrient agar and on a plate whose agar contained chloramphenicol.
  • After overnight incubation at 37 C, a lawn of bacteria was found on the plain nutrient agar plate.
  • No lawn and no visible signs of bacteria appeared on the plate with tetracycline.
• An equivalent number of bacteria, chemically treated to make them competen,t were treated with 1 nanog of pSC101DNA (ref), a plasmid DNA, and then plated on a similar pair of plates.
  • Again a lawn was seen on the nutrient agar alone plate, after overnight incubation.
  • On the tetracycline containing plate about 200 colonies of bacteria were evident.
• This use of antibiotic containing plates to retrieve transformed bacteria, but not untransformmed ones, is called selection.

Interpretations

• The host E. coli cells are sensitive to inhibition of growth by tetracycline.
• The plasmid pSC101 contain a gene that confers resistance to tetracycline on the E. coli strain.
• The presence of tetracycline in the growth medium selectively allows bacteria containing pSC101 to grow.
• The efficiency of transformation is 2 x 10⁵ transformants per microg of DNA (in this case one microg DNA is approximately equal to 10⁸ molecules).
• Separated populations of E. coli, for example, colonies, may be viusally screened for the presence or absence of certain features. Such screening differs from selection in that, for screening, all populations must grow while for selection only the desired ones grow.

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Further information

• In work with E. coli, bacteriostatic or bactericidal agents are usually used for selection. Examples of these agents are:
  • inhbitors of peptidoglycan (cell wall) synthesis (pneicillin, ampicillin),
  • inhibitors of translation (protein synthesis) (kanamycin, chloramphenicol, tetracycline, streptomycin),
  • inhibitors of transcription (RNA synthesis) (rifampin, rifampicin),
  • DNA gyrase inhibitors (coumarin, brefeldin).
• Genes encoding proteins conferring resistance to the antibacterial agents produce resistance through their encoded proteins in different ways:
  • Some hydrolyze the antibiotic (beta-lactamase);
  • Some phosphorylate the antibiotic (neomycin phosphotransferase);
  • Some acetylate the antibioitc (chloramphenicol acetyl transferase);
  • some are pumps that export the antibiotic (tetracycline transporter),
  • some are mutated proteins whose mutation makes their activity insensitive to the antibiotic (RNA polymerase, DNA gyrase, dihydrofolate reductase, inter alia).
• In other organisms, such as yeast, the selection can be accomplished by omission of an essential component from the nutrient solution. The transforming DNA will have a gene for an enzyme whose introduction will lead to synthesis of the missing compound. For example,
  • yeast cells lacking a gene for an enzyme in the biosynthesis pathway of the amino acid leucine are used as recipients in transformation.
  • The transforming vector has a copy of the functioning gene and can therefore be selected for in media lacking leucine.
• There are many purposes for molecular cloning.