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Bacteriophage Vectors
Bacteriophages are viruses that attack bacteria. Most phages lyse the bacterial cells they infect (lytic phages). But many others can choose to follow either a lytic or a lysogenic cycle; in the latter situation, the phage chromosome integrates into the bacterial chromosome and multiplies with the latter as prophage (temperate or lysogenic phages). The prophage may dissociate from the bacterial chromosome and follow the lytic cycle.
Several bacteriophage are used as cloning vectors, the most commonly used E. coli phages being λ (lambda) and M13 phages. Plasmid vectors have to be introduced into bacterial cells which are then cloned and selected for the recovery of recombinant vectors. In contrast, the phage vectors are directly tested to an appropriate bacterial lawn (a continuous bacterial-free zone in the bacterial lawn). Phage vectors present two advantages over plasmid vectors. (1) They are more efficient than plasmids for cloning of large DNA fragments; the largest cloned insert size in a λ vector is just over 24 kb, while that for plasmid vectors it is less than 15 kb. In addition, (2) it is easier to screen a large number of phage plaques than bacterial colonies for the identification of recombinant vectors.
λ Phage Vectors
The λ genome (total 48,502 bp) contains an origin of replication; genes for head and tail proteins and enzymes for DNA replication, lysis and lysogeny; and single-stranded protruding cohesive ends of 12 bases (5’ GGGCGGCGACCT; the other end is complementary to it; i.e. CCCGCCGCTGGA 5’). The λ genome remains linear in the phage head, but within E. coli cells the two cohesive ends anneal to form a circular molecule necessary for replication. The sealed cohesive ends are called cos sites which are the sites of cleavage during and are necessary for packaging of the mature phage DNA into phage heads. The λ DNA must be larger than 38 kb and smaller than 52 kb to be packaged into phage particles. The genes for lysogeny are located in the segment between 20 and 38 kb; the whole or a part of this segment is deleted to create λ vectors to (1) accommodate larger DNA inserts and (2) to ensure that the recombinant phage is always lytic.
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Several bacteriophage are used as cloning vectors, the most commonly used E. coli phages being λ (lambda) and M13 phages. Plasmid vectors have to be introduced into bacterial cells which are then cloned and selected for the recovery of recombinant vectors. In contrast, the phage vectors are directly tested to an appropriate bacterial lawn (a continuous bacterial-free zone in the bacterial lawn). Phage vectors present two advantages over plasmid vectors. (1) They are more efficient than plasmids for cloning of large DNA fragments; the largest cloned insert size in a λ vector is just over 24 kb, while that for plasmid vectors it is less than 15 kb. In addition, (2) it is easier to screen a large number of phage plaques than bacterial colonies for the identification of recombinant vectors.
λ Phage Vectors
The λ genome (total 48,502 bp) contains an origin of replication; genes for head and tail proteins and enzymes for DNA replication, lysis and lysogeny; and single-stranded protruding cohesive ends of 12 bases (5’ GGGCGGCGACCT; the other end is complementary to it; i.e. CCCGCCGCTGGA 5’). The λ genome remains linear in the phage head, but within E. coli cells the two cohesive ends anneal to form a circular molecule necessary for replication. The sealed cohesive ends are called cos sites which are the sites of cleavage during and are necessary for packaging of the mature phage DNA into phage heads. The λ DNA must be larger than 38 kb and smaller than 52 kb to be packaged into phage particles. The genes for lysogeny are located in the segment between 20 and 38 kb; the whole or a part of this segment is deleted to create λ vectors to (1) accommodate larger DNA inserts and (2) to ensure that the recombinant phage is always lytic.
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