Question

Bacteriophage Lambda phage have the ability to survive either as a lysogen w/in a bacterial host or by lytic growth. Lytic growth involves replication of the viral DNA manyfold, assembly of bacteriophage particles, and cell lysis to release the new infectious bacteriophage particles. The bacteriophage particles can each package one entire viral genome of ~50Kb but not much more DNA than that. Use your knowledge of the regulation of the lambda gene expression to design a lambda cloning vector. The cloning vector should be replicated manyfold, and assemble into infectious viral particles that are released by lysis of the host bacterial cell. Hint: think about what genes are necessary for lysogenic growth and dispensable for lytic growth. These are the genes that you can replace with your DNA of interest.

Answer 1

The Lambda phage genome of 50 Kb contains genes for capsid as well as for other proteins involved in lytic and lysogenic pathways.It can not harbour any more additional DNA.As such inorder to convert Lambda phade genome into a vector some of the genes of its genome which are not required for lytic pathway can be deleted.And in place of such genes, foreign DNA of interest can be incorporated into Lambda phage DNA to generrate a cloning vector.The Lambda vector is prepared in the following steps.

1.Lambda DNA is first treated with a restriction enzyme to bproduce Lambda vector arms. that have sticky ends and all the necessary genes required for lytic pathway.In this step nonessential region present in middle of the genome is removed.

2.The DNA of interest is extracted and also treated with the same restriction enzyme to produce fragments of 20kb length with sticky ends.complementary to the sticky ends of vector arms.

3. Lamda vector arms and the DNA fragments of interest aremixed in equal amounts.The complementary sticky ends of DNA fragments and vector arms hybridize and are ligated by DNA ligas.As a result the molecule formed is recombinant vector molecule.