7.14C: Hosts for Cloning Vectors
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The majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) as the host.
Describe the features of a typical cloning vector
- E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.
- If the DNA to be cloned is exceptionally large, then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.
- Specialized applications may call for specialized host -vector systems.
- Escherichia coli: Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms).
- plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.
- molecular cloning: a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.
A very large number of host organisms and molecular cloning vectors are in use, but the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.
Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an expression vector is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example transfer of DNA from bacteria to plants), then a multiple host range vector (also termed shuttle vector) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.
Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility–(1) an origin of DNA replication is necessary for the vector (and recombinant sequences linked to it) to replicate inside the host organism, (2) one or more unique restriction endonuclease recognition sites that serves as sites where foreign DNA may be introduced, (3) a selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences, and (4) an additional gene that can be used for screening which cells contain foreign DNA.