Bacteriophage Vectors Lamda Phage Vector

Bacteriophage Vectors Lamda Phage Vector

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  Page 1 of5 Introduction to Bacteriophage Vectors • A bacteriophage is a type of virus that infects bacteria. In genetic engineering, bacteriophages are often used as vectors for cloning genes, due to their ability to efficiently introduce foreign DNA into bacterial cells. • Bacteriophage vectors utilize the natural ability of bacteriophages to infect and deliver genetic material to bacteria, making them ideal for gene transfer, cloning, and protein expression. • They are often used in molecular biology, recombinant DNA technology, and other research applications. Types of Bacteriophage Vectors: 1. Lambda (λ) Phage Vectors: o Lambda phages are commonly used for cloning large fragments of DNA. o Lambda vectors can carry large insertions (up to 20 kb) and are ideal for genomic libraries.
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  Page 2 of5 Lamda phage in Cloning A lambda phage is also a vector used for DNA cloning. It is a virus containing double-stranded phage DNA that can is introduced into E. Coli for replication. The ssDNA is linear in Virus, become circular in Bacteria Host. Lamda genome is 49 kb in length. Only 30kb is required for Lytic cycle. So we can insert up to 19kb of foreign DNA/GOI. It has packaging capacity of 78-100%. Mutant lambda phages have been created that makes cloning easier. One example is the lambda-gt-lambda-beta phage. It contains two EcoRI cleaves sites. The middle section of the phage DNA is removed by restriction digestion. The two remaining arms of the DNA cannot be packaged in to lambda virion because it is too small.
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  Page 3 of5 Instead, a foreign piece of target DNA is joined to the two arms with ligase, which replaces the middle section that was removed. The addition of the foreign DNA allows the phage DNA to be long enough to be packed into a virion. The packaged lambda virion can then be inserted into a host cell and then replaced through the lytic or lysogenic pathway. 1. Attachment to Bacteria (Adsorption): • The λ phage binds specifically to receptors on the surface of E. coli, typically the maltose transporter (LamB) protein. • The phage tail fibers attach to the receptor, anchoring the phage to the bacterial surface. 2. Injection of Phage DNA: • After attachment, the phage contracts its tail sheath, punctures the bacterial outer membrane, and injects its double-stranded DNA (dsDNA) into the bacterial cytoplasm. 3. Deciding Between Lytic or Lysogenic Pathway: Once inside the host cell, the phage DNA takes one of two pathways: A. Lysogenic Cycle (Dormant State): • The phage integrates its DNA into the host's genome at a specific site (the attB site) using the phage-encoded integrase enzyme. • Once integrated, the phage DNA is called a prophage and is replicated passively as part of the host chromosome during bacterial cell division. • During this phase, phage genes for lysis and replication are repressed by the cI repressor protein, which prevents activation of the lytic genes. B. Lytic Cycle (Active Replication): • If conditions favor phage replication (e.g., bacterial stress or DNA damage), the repressor (cI) is inactivated, and the lytic cycle begins.
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  Page 4 of5 • The phage DNA is expressed, and viral components (capsid proteins, tail fibers, etc.) are produced. • The host DNA is degraded to provide nucleotides for phage genome replication. 4. Phage Genome Replication: • The phage DNA is replicated using the host's replication machinery: o Theta replication: Starts initially, producing a few copies of circular phage DNA. o Rolling-circle replication: Produces long linear DNA molecules (concatemers) containing multiple phage genomes. 5. Assembly of New Phages: • Phage capsid proteins and tail structures are synthesized and assembled. • The concatemer DNA is cleaved into individual genome-length fragments and packaged into the capsids. 6. Lysis and Release of Progeny Phages: • The phage produces enzymes like holin and endolysin to break down the bacterial cell wall. • The host cell bursts (lysis), releasing hundreds of new λ phages into the environment to infect other bacteria. 2. M13 Phage Vectors: o M13 is a filamentous bacteriophage, often used as a cloning vector for smaller DNA fragments (around 6-10 kb). o Unlike lambda phages, M13 does not lyse the host cell, making it useful for cloning without killing the host. 3. T7 Phage Vectors: o T7 phages are used for high-efficiency protein expression and cloning of smaller inserts (up to 10 kb).
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  Page 5 of5 Advantages of Bacteriophage Vectors: • High efficiency: Phages naturally infect bacterial cells, allowing high- efficiency DNA delivery. • Capacity for large DNA inserts: Certain phage vectors, like Lambda, can carry large DNA fragments. • Cloning and expression capabilities: Many phage vectors can be engineered to express proteins from inserted genes. • Stability: Phages are relatively stable and can be maintained easily in bacterial cultures.