Bacteriophage vectors, Biology

Bacteriophage Vectors

Bacteriophages are viruses that attack bacteria. Mainly phage lyses the bacterial cells they infect (lytic phages). But many others may choose to follow either lysogenic or a lytic cycle; in the second situation, the phage chromosome integrates into the bacterial chromosome and multiplies with the latter as prophage (temperate or lysogenic phages). The prophage can dissociate from the bacterial chromosome and follow the lytic cycle.
 
Many  bacteriophage are used as cloning vectors, the most generally used E. coli phages being λ (lambda) and M13 phages. Plasmid vectors have to be introduced into bacterial cells which are then cloned and choose for the recovery of recombinant vectors. In contrast, the phage vectors are sprightly tested to an suitable bacterial lawn (a constant bacterial-free zone in the bacterial lawn). Phage vectors show  two advantages over plasmid vectors. (1) it is easier to screen a large number of phage plaques than bacterial colonies for the identification of recombinant vectors.(2) 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 λ Phage Vectors

The λ genome (total 48,502 bp) contains an source of replication; genes for head and tail  enzymes and proteins for DNA replication lysogeny and lysis; and single-stranded protruding cohesive ends of 12 bases (5' GGGCGGCGACCT; the other end is opposite 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 make a circular molecule essential for replication. The sealed cohesive ends are known cos sites that  are the sites of cleavage during and are essential for packaging of the mature phage DNA into phage heads. The λ DNA need to be larger than 38 kb and smaller than 52 kbto be packaged into phage particles. The genes for lysogeny are situated in the segment among 20 and 38 kb; the complete 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.

Posted Date: 10/2/2012 1:23:55 AM | Location : United States







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