Phage Cloning Vectors

Cloning vectors derived from λ bacteriophage are commonly used as it provides an approximately 16-fold advantage in cloning efficiency in comparison with the most efficient plasmid cloning vectors. ^[5] Phage λ is a linear double-stranded phage approximately 49 kb in length. It infects E. coli with great efficiency by injecting its DNA through the cell membrane. ^[5]  In the wild-type phage λ the DNA follows one of two possible modes of replication. ^[5] Firstly the DNA may either become stably integrated into the E. coli chromosome where it lies dormant until a signal triggers its excision. This is termed the lysogenic life cycle. ^[5] Alternatively, it may follow a lytic life cycle where the DNA is replicated upon entry to the cell, phage head and tail proteins synthesized rapidly and new functional phage assembled. The phage is subsequently released from the cell by lysing the cell membrane and infects further E. coli cells nearby. ^[5]  Phage may be replicated very and result of which are concatemers of many phage genomes which are cleaved at the cos sites and inserted into newly formed phage protein heads. Much use of phage λ has been made in the production of gene libraries. ^[5]

Feature of  λ vector:

1. The  genes related in function are clustered together. So it allows swiching on or off a group of genes rather than individual.
2. Conformation of DNA molecule. It is linear molecule consists of two complementary strands of DNA. The complementary strand are called cos site.

Two types of λ phage vectors have been developed:

λinsertion vectors: Large segment of non essential region has been deleted and the two arms are ligated together. An insertion vector possesses at least one unique restriction site into which new DNA can be inserted.[4]

Two popular insertion vectors are  λgt10 and λcharon16A.

λreplacement vectors: It has two recognition sites for restriction endonuclease. These sites flank a segment of DNA that is replaced by DNA to be cloned.[4] 

Two popular insertion vectors are λEMBL and λZap

λZap is a commercially produced cloning vector that includes unique cloning sites clustered into a multiple site (MCS). MCS is located within a lacZ region providing a blue/white screening system based on insertional inactivation. It is also possible to express foreign cloned DNA from this vector. ^[5]  This is a very useful feature of some vectors since it is then possible to screen for protein product rather than the DNA inserted into the vector. This screening is therefore undertaken with antibody probes directed against the protein of interest. Other features that make this a useful cloning vector are the ability to produce RNA transcripts termed cRNA or riboprobes. ^[5]  This is possible because two promoters for RNA polymerase enzymes exist in the vector, a T7 and a T3 promoter which flank the MCS. ^[5]

Improved phage vectors were developed for:

• Increasing the capacity of foreign DNA fragment.
• Devising positively selecting recombinant formation.
• Allowing RNA probe to be conveniently prepared.
• Development of vectors for the insertion of eukarykaryotic cDNA.

 Image courtesy: fig 6.13, Principles and Techniques of Biochemistry and Molecular Biology , Wilson Keith and Walker John, seventh edition, Cambridge university press, 2010

                                                                                    Previous           Home             Next