Bacteriophage Lambda Vectors

Bacteriophage lambda, which infects E. coli, has been widely used as a cloning vector. As described in Ch. 18, lambda is a well-characterized virus with both lytic and lysogenic alternatives to its life cycle. Although lambda DNA circularizes for replication and insertion into the E. coli chromosome, the DNA inside the phage particle is linear (Fig. 22.18). At each end are complementary 12 bp long overhangs known as cos sequences (cohesive ends). Once inside the E. coli host cell, these pair up and the cohesive ends are ligated together by host enzymes forming the circular version of the lambda genome.

Only DNA molecules of between 37 and 52 kb can be stably packaged into the head of the lambda particle. Small fragments of extra DNA may be inserted into the lambda genome without preventing packaging. However, to accommodate longer inserts it is necessary to remove some of the lambda genome. The left hand region has essential genes for the structural proteins and the right hand region has genes for replication and lysis.The middle region (~15 kb) of the lambda genome is non-essential and may be replaced with approximately 23 kb of foreign DNA (Fig. 22.19). Since the middle region of lambda has the genes for integration and recombination, such lambda replacement vectors cannot integrate into the host chromosome and form lysogens by bacteriophage lambda Virus of E. coli with both lytic and lysogenic alternatives to its life cycle, which is widely used as a cloning vector Cen sequence See centromere sequence cos sequences (lambda cohesive ends) Complementary 12 bp long overhangs found at each end of the linear form of the lambda genome

FIGURE 22.18 Lambda-Linear and Circular Genomes

In the lambda phage particle, the genome is a linear DNA molecule with two cos sequences at each end. After the phage injects its DNA into the bacterial host, the DNA circularizes. The two cohesive ends base pair and are ligated together by bacterial enzymes so forming a circle.

Linear

(inside virus Q particle)

Circularization

Circular

(after infection of bacterial cell)

Attachment site

Attachment site

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