M13 VECTOR in Microbial Biotechnology.pptx

M13 Bacteriophage

1. VIVEKANANDHA ARTS AND SCIENCE COLLEGE FOR WOMEN VEERCHIPALYAM-637303,SANKAGIRI,SALEM Dt.,TAMILNADU,INDIA. AFFILIATED TO PERIYAR UNIVERSITY,SALEM;RECOGNISED UNDER SECTION 2(F)&12(B) OF THE UGC ACT 1956) SUBJECT INCHARGE; Dr.R. Pandiyan , Assistant professor, Department of Microbiology, VIAAS,Sankagiri. DEPARTMENT OF MICROBIOLOGY SUBJECT : MICROBIAL BIOTECNOLOGY SUBMITTED BY; KANIMOZHI.I.S., III B.SC.MICROBIOLOGY, DEPARTMENT OF MICROBIOLGY, VIAAS,Sankagiri. TITLE : M13

2. MI3 VECTOR

3. MI3 VECTOR M13 is one of the Ff phages , a member of the family filamentous bacteriophage . Ff phages are composed of circular single-stranded DNA (ssDNA), which in the case of the m13 phage is 6407 nucleotides long and is encapsulated in approximately 2700 copies of the major coat protein p8, and capped with about 5 copies each of four different minor coat proteins . The minor coat protein p3 attaches to the receptor at the tip of the F pilus of the host Escherichia coli. The life cycle is relatively short, with the early phage progeny exiting the cell ten minutes after infection.

4. Ff phages are chronic phage, releasing their progeny without killing the host cells. The infection causes turbid plaques in E. coli lawns, of intermediate opacity in comparison to regular lysis plaques. However, a decrease in the rate of cell growth is seen in the infected cells. The replicative form of M13 is circular double-stranded DNA similar to plasmids that are used for many recombinant DNA processes, and the virus has also been used for phage display, directed evolution, nanostructures and nanotechnology applications. Example:plasmid,phage\virus

5. TYPES OF VECTORS 1. M13mp Series: M13mp18: Contains a multiple cloning site (MCS) within the lacZ’ gene, allowing for blue/white screening of recombinant clones1. M13mp19: Similar to M13mp18 but with a different orientation of the MCS1. 2. M13KO7: M13KO7: A helper phage used to produce single-stranded DNA from phagemid vectors. It carries a kanamycin resistance gene .

6. 3. M13KE:M13KE: Used for phage display, allowing the presentation of peptides or proteins on the phage surface . APPLICATION OF M13 VECTORS: M13 vectors have a variety of applications in molecular biology and biotechnology. 1. DNA Sequencing: Single-Stranded DNA Production: M13 vectors are used to produce single-stranded DNA, which is essential for Sanger sequencing 2. Cloning and Mutagenesis: Cloning Vectors: M13 vectors can carry foreign DNA fragments, making them useful for cloning and genetic manipulation2. Site-Directed Mutagenesis: The single-stranded DNA form of M13 is ideal for introducing specific mutations into a DN3.

7. 3. Phage Display: Protein and Peptide Display: M13 vectors are used in phage display technology to present peptides or proteins on the surface of the phage. This is useful for identifying protein-protein interactions, antibody engineering, and drug discovery . 4. Nanotechnology: Nanostructures: M13 bacteriophages can be engineered to form nanostructures and nanomaterials for various applications, including biosensing and materials science4

8. STRUCTURE OF M13 VECTORS: G enome: Type: Circular single-stranded DNA (ssDNA). Length: Approximately 6407 nucleotides . Capsid Proteins: Major Coat Protein (pVIII): Function: Forms the bulk of the filamentous phage coat. Quantity: Around 2700 copies per phage.

9. Minor Coat Proteins: pIII and pVI: Located at one end of the phage, involved in host cell attachment. pVII and pIX: Located at the opposite end, involved in phage assembly . Phage Structure: Shape: Filamentous, resembling a long, thin rod. Dimensions: The length of the phage is flexible and depends on the size of the encapsulated DNA.

10. REPLICATION OF M13 VECTORS Infection: The M13 phage attaches to the F pilus of E. coli using its pIII protein. The single-stranded DNA (ssDNA) genome of the phage enters the host cell. Conversion to Double-Stranded DNA: Inside the host cell, the ssDNA is converted into a double-stranded replicative form (RF) by host enzymes. This RF serves as a template for transcription and replication.

11. Rolling Circle Replication: The RF undergoes rolling circle replication, producing multiple copies of ssDNA. The gene II protein introduces a nick in the positive strand of the RF, initiating the replication process. DNA polymerase III extends the positive strand, displacing the old strand, which is then circularized to form new ssDNA genomes . Packaging and Assembly: The newly synthesized ssDNA is coated with the gene V protein to prevent it from being converted back to RF. The ssDNA is then packaged into new phage particles as it is extruded through the host cell membrane, where it is coated with the major coat protein (pVIII).

12. Re lease: The assembled phage particles are released from the host cell without lysing it, allowing continuous production of phages. This efficient replication mechanism makes M13 vectors highly useful for various applications, including cloning, sequencing, and phage display. ADVANTAGE: M13 vectors offer several advantages that make them valuable tools in molecular biology and biotechnology: 1. Production of Single-Stranded DNA: Sequencing: M13 vectors are excellent for producing single-stranded DNA, which is crucial for Sanger sequencing. Mutagenesis: The single-stranded form is ideal for site-directed mutagenesis, allowing precise genetic modifications.

13. 2. Phage Display: Protein and Peptide Display: M13 vectors are widely used in phage display technology to present peptides or proteins on the phage surface. This is useful for studying protein-protein interactions, antibody engineering, and drug discovery . 3. Cloning and Subcloning: Versatility: M13 vectors have multiple cloning sites (MCS) that facilitate the insertion of foreign DNA. They can be used to clone and subclone DNA fragments efficiently. Blue/White Screening: The presence of the lacZ’ gene allows for blue/white screening, making it easy to identify recombinant clones.

14. 4. Continuous Phage Production: Non-Lytic Cycle: M13 phages do not lyse the host cell, allowing continuous production of phages and making them suitable for long-term experiments . 5. Nanotechnology Applications: Nanostructures: M13 bacteriophages can be engineered to form nanostructures and nanomaterials, which have applications in biosensing and materials science. These advantages highlight the versatility and utility of M13 vectors in various scientific and technological applications. DISADVANTAGE: 1. Size Limitation: Cloning Capacity: M13 vectors can only accommodate relatively small DNA inserts, typically up to 2 kb. This limits their use for cloning larger DNA fragments.

15. 2. Low Yield: DNA Yield: The yield of DNA from M13 vectors is generally lower compared to other vectors like plasmids. This can be a drawback when large amounts of DNA are required. 3. Slow Growth: Host Cell Growth: M13 infection does not lyse the host cell but slows its growth, leading to turbid plaques rather than clear ones. This can complicate plaque assays and other experiments. 4. Toxicity: Phage Proteins: High concentrations of phage proteins can be toxic to the host cells. This can affect the efficiency of phage production and the health of the bacterial culture.

16. 5. Limited Amplification: Genome Amplification: M13 vectors cannot amplify phage genome numbers as efficiently as other vectors. This can be a limitation for certain applications requiring high phage titers. These disadvantages should be considered when choosing a vector for specific applications.

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