2 µm plasmid 

Image source:  Figure 7.1, Gene Cloning & DNA Analysis- An Introduction, T.A.Brown. fifth edition, blackwell publishing, 2010

 The 2 µm plasmid is an excellent basis for a cloning vector. ^[4] It is 6 kb in size and has a high copy number of between 70 and 200.

Replication makes use of a plasmid origin, several enzymes provided by the host cell, and the proteins coded by the REP1 and REP2 genes carried by the plasmid. ^[4]

However, all is not perfectly straightforward in using the 2 µm plasmid as a cloning vector. ^[4]

First, there is the question of a selectable marker. ^[4] For this purpose a normal yeast gene is used, generally one that codes for an enzyme involved in amino acid biosynthesis. An example is the gene LEU2, which codes for β-isopropyl-malate dehydrogenase, one of the enzymes involved in the conversion of pyruvic acid to leucine. ^[4]

In order to use LEU2 as a selectable marker, a special kind of host organism is needed. The host must be an auxotrophic mutant that has a non-functional LEU2 gene. ^[4]

Such a leu2− yeast is unable to synthesize leucine and can survive only if this amino acid is supplied as a nutrient in the growth medium. Selection is possible because transformants contain a plasmid-borne copy of the LEU2 gene, and so are able to grow in the absence of the amino acid. ^[4] In a cloning experiment, cells are plated out onto minimal medium, which contains no added amino acids. Only transformed cells are able to survive and form colonies and thus are easily differentiated.

                                                                                     Previous          Home          Next