Recombinant Dna Technology: M 13 Bacteriophage

WEST BENGAL UNIVERCITY OF TECHNOLOGY DEPARMENT OF BIOTECHNOLOGY
RECOMBINANT DNA TECHNOLOGY

M 13 BACTERIOPHAGE
PRASHANT BAJPAI M.TECH BIOTECH 1ST YEAR
M13 BACTERIOPHAGE
Introduction: M13 Bacteriophage is a single-stranded filamentous DNA bacteriophage. This phage infects only male bacterial cells, after attachment to the male-specific pilus (F pilus, present in suitable host cells with the genotype E. coli F'). Upon entering the cell, the phage is stripped of its protein coat, and the single-stranded DNA is converted into a double stranded replicative form (RF). This is followed by DNA replication and assembly of new particles. Roughly 200 phage particles are produced per infected cell, per generation. M13 and the other filamentous DNA phages are unusual in that they are released from the infected cell, without causing the death of the cell. M13 bacteriophage causes chronic infections, and infected cells continue to grow and divide, although at a lower rate than normal. Thus, phage plaques of M13 are seen as areas of reduced growth in the bacterial lawn (i.e., clear zones of bacterial lysis do not occur). Two other unusual feature of M13 are: (i) Stability to extremes of pH and temperature (pH as low as ~pH 3, and temperature up to
o
55 C) and (ii) its unusual shape and length (it is rod-shaped, and approx. 6 nm in diameter and 860 nm in length). M13 has historically been used a cloning vehicle, and it has also been widely used for DNA sequencing (this is because the viral particle contains single-stranded DNA, which is an ideal template for DNA sequencing).
Structure of M13 Bacteriphage:
Structure and membrane embedding of M13 coat protein
The phage is a long thread-shaped particle and has a diameter of 6.5 nm and is 900 nm long. The flexible filament contains a circular, single-stranded viral DNA genome, having 6,407 nucleotides, that is protected by a long cylindrical protein coat. This coat is made up of approximately 2,700 copies of the major coat protein (gp8; Fig. 2) capped by minor coat proteins (35 copies each) gp3 and gp6, or gp7 and gp9 at both ends (Webster and Lopez 1985). The major coat proteins form a tube around the viral DNA, in an overlapping helical array.They are oriented such that the N terminus is located at the outside of the coat and the C terminus interacts with the DNA at the inside of the coat. The hydrophobic domain of the major coat protein is located in the central part of the protein, and it interlocks the coat protein with its neighbouring coat proteins in the viral particle. M13 Bacteriophage Replication and Selection:
Phage infection takes place, first by binding to sex pili through adsorption proteins located at attachment end that means they infect only male bacterial cells. Then it is through pili, phage DNA enters into the cell. Infection of bacteria without sex pili also takes place, but with very low frequency. Many speculations are in air about the mechanism by which the DNA enters. According to one hypothesis, DNA enters along the groove of pili. Second speculation is that the phage is guided by the pilus to an outer membrane receptor through which DNA enters the cell. The third group thinks, with adsorption to the tip of the pilus, the pilus undergoes deploymerization from membrane side that is from its base where it has anchored to the membrane, with attachment of the gp6 and gp3 to the pilius the gp8 capsid proteins undergo conformational change in their helical structure. With more penetration they undergo more changes, by the time the DNA enters the proteins undergo full conformational changes, thus the DNA enters into the cytoplasm and gp8 capsid proteins enter into bacterial plasmamembrane. Once inside the cell the single stranded molecule acts as the template for the synthesis of a complementary strand, resulting in normal double stranded DNA. This molecule is not inserted in to bacterial genome but instead replicates until over 1000 copies are present in the cell. When the bacterium divides, each daughter receives copies of the phage genome. This continues to
replicate thereby maintaining its overall numbers per cell. The new phage particles are continuously assembled and released. Selection: The selection strategy for recombinant DNA utilize a part of the E.Coli Lac Z gene (lac Za). The unchanged M13 vector produce blue plaques on the lawn of an appropriate E. Coli strain. The recombinant DNA produce colorless plaques which are readily identified. A 42bp poly linker sequence having several unique restriction sites is placed within the lac Za that encode amino terminal portion of the enzyme -galactosidase. When a DNA insert is placed anywhere within the poly linker sequence the formation of amino-terminal part of the enzyme is prevented. Applications: M13 bacteriophage has application in: Sequencing Mutagenesis Probes lambda-ZAP subcloning phage display libraries
Phage Display Library: Examples of phage display vector approaches
Phage display technology involves the expression of exogenous proteins, peptides or peptide libraries on the surface of bacteriophage. The most commonly used phage for this work is M13 (or other filamentous phage). However, T7-based display systems have also become available commercially and offer a useful alternative approach. Applications of this technology include the identification of novel peptides with the ability to bind to defined target molecules (e.g., cell surface receptors or enzymes), and the identification of human monoclonal antibodies with defined reactivity against specific targets (in this case, antibody phage display libraries are used). Other examples of the uses of this technology include the use of recombinant bacteriophage displaying antigens from infectious disease agents as candidate vaccines, to confer immune responses against the encoded peptides or proteins. Advantages of the technology include its simplicity, ease-of-use, low cost, safety and immunogenicity (for vaccine studies). In the case of antibody display libraries, phagemid vectors are typically used (since they have a very large insert capacity) and the vector system is generally selected such that there is an average of perhaps 0.1 copies of antibody per phage. Thus, most phage lacks any displayed antibody, and almost no phage will display any more than a single antibody molecule.

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WEST BENGAL UNIVERCITY OF TECHNOLOGY DEPARMENT OF BIOTECHNOLOGY

RECOMBINANT DNA TECHNOLOGY

PRASHANT BAJPAI M.TECH BIOTECH 1ST YEAR

Structure and membrane embedding of M13 coat protein

Phage Display Library: Examples of phage display vector approaches

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