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Cloning VectorsComments

Cloning Vectors

Vector DNA (or just vector, for short) is a DNA molecule which exists within a host cell, is not part of the gnome and which replicates independent of the host genome. Cells can be induced to uptake a manmade vector containing genes of interest (a process called transformation) so that the functions of those genes may be studied.

Also, genes of interest can be inserted into rapidly-replicating vectors; after allowing for replication, vast amounts of the inserted gene can be isolated. Similarly, transformed cells can produce large amounts of a useful protein or chemical encoded by the vector. Genes inserted into a vector (or insert, for short) can be of any origin, and the ligation (binding) of DNA from different sources is known as recombinant DNA technology.

Common vectors are plasmids, phages, cosmids, bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC).

Plasmids

Circular double-stranded vectors are called plasmids. Plasmids were first discovered in bacteria, and carried antibiotic resistance genes from one bacterium to another. Since then, plasmids have been engineered which are small and contain three critical components:

  1. An origin of replication,
  2. One or more selectable markers (aka indicator genes).
  3. One or more restriction sites (aka cloning sites).

Selectable markers allow transformants (aka recombinants, bacteria which contain the vector) to be identified from non-transformed cells. For example, a penicillin-susceptible culture could be transformed with a plasmid conferring penicillin resistance; subsequently, penicillin could be added to kill any cells that did not uptake the plasmid DNA. Common indicator genes include TetR (tetracycline resistance) and lacZ (containing a cloning site, only non-blue colonies are transformed).

Restriction sites allow foreign genes to get inserted into the plasmid. First, the plasmid is digested by a restriction enzyme; second, the plasmid fragments are mixed with genes of interest; and third, ligase is added so that the plasmid fragments and the genes recombine. While some DNA might recombine without the insert (or in the wrong order), sheer quantity means that enough plasmids and genes recombine correctly. A polylinker is a sequence containing restriction sites for multiple different restriction enzymes.

Bacterial Artificial Chromosome (BAC)

Bacterial artificial chromosomes are used to genetically engineer prokaryotes. They are introduced into the cell generally by transformation or (less often) by transduction.

  • Origin of replication
  • Genes repressing high copy number
  • Reporter gene (i.e., antibiotic resistance or lacZ)
  • Promoter in front of reporter gene (directionality and strength)
  • Restriction/Multiple-Cloning Site (MCS) within reporter
  • ChloramphenicolR
  • Circular plasmid based on F plasmid
  • SapB and SapA keep copy number lower