Phagemid
In molecular biology, a phagemid or phasmid is a plasmid which has an origin of replication for the single-stranded replication of f1 phages .
properties
Phagemids were developed in the early 1980s and serve as vectors for DNA cloning .
A phagemid can normally be propagated as a plasmid - since it has a corresponding origin of replication ( ori ) - and is then double-stranded. On the other hand, due to the f1 intergenic region, which contains all cis-acting elements for replication ( ori ) and for packaging , the phagemid can also be propagated with the help of a helper phage . To do this, the bacterial culture must be infected with a helper phage, because this is the only way to create the necessary viral components for single-stranded replication in the host celland packaging the phagemid DNA into a phage particle. M13 phages or f1 phages are often used for this purpose. The helper phages are usually chosen so that their own DNA is incorporated into new phage particles less effectively than the phagemid. Consequently, new phage contain mainly the phagemid and not the helper phage DNA. In the phages, the DNA is single-stranded due to the selected f1 ori .
Infected bacterial cultures are centrifuged overnight to collect the phages. The plasmids can be isolated from the bacterial pellet. The phages are secreted and can be enriched in the supernatants by means of polyethylene glycol (PEG) precipitation (150 μl 20% PEG-8000 / 2.5 M NaCl per 1 ml). The single-stranded DNA can be purified from the precipitate by means of phenol-chloroform extraction and ethanol precipitation.
Many plasmids used today, such as pBluescript II , contain an f1 ori and are therefore phagemids. Like a plasmid, a phagemid can be used to clone fragments of DNA .
Individual evidence
- ^ A b Analysis of Genes and Genomes, John Wiley & Sons, 2004, p. 140, Google Books
- ^ H. Qi, H. Lu, HJ Qiu, V. Petrenko, A. Liu: Phagemid vectors for phage display: properties, characteristics and construction. In: J Mol Biol. (2012), Volume 417, No. 3, pp. 129-143. doi : 10.1016 / j.jmb.2012.01.038 . PMID 22310045 .