<h5 id="21-two-key-components-of-recombinant-dna-technology">2.1 Two Key Components of Recombinant DNA Technology</h5> <ul> <li>Recombinant DNA (rDNA) technology involves joining two different DNA molecules for various applications.</li> <li>It consists of two major steps: <ol> <li>Isolation of the desired DNA molecule (insert or target gene) from its source.</li> </ol> </li> </ul> <p>======</p> <h5 id="22-host">2.2 Host</h5> <ul> <li>A wide variety of organisms, both prokaryotic and eukaryotic, are used as hosts for gene cloning.</li> <li>Prokaryotic hosts are preferred as they allow easy entry of rDNA, supply required enzymes and proteins for replication, and do not degrade the recombinant DNA.</li> <li>Escherichia coli (E. coli) is the most extensively used prokaryotic host, especially the K12 strain, which is known for its rapid reproduction and growth.</li> <li>Other prokaryotic hosts like Bacillus subtilis are used when the aim is secretion of a protein encoded by a cloned gene.</li> <li>Yeast is the most widely used eukaryotic host, due to its ability to supply all required enzymes and proteins for smooth replication of the vector DNA along with the insert.</li> </ul> <p>======</p> <h5 id="23-vector">2.3 Vector</h5> <ul> <li>DNA vectors should be small in size and capable of integrating a large insert for easy incorporation into the host cell.</li> <li>They must have an origin of replication (ori) for autonomous replication inside the host organism.</li> <li>Vectors need to have unique restriction sites to avoid fragmentation into several pieces.</li> <li>A selectable marker, such as antibiotic resistance (e.g., tetracycline or ampicillin), is required for screening transformants.</li> <li>Various kinds of eukaryotic and prokaryotic hosts and vectors are available for gene cloning.</li> </ul> <p>======</p> <h5 id="231-plasmid-as-a-vector">2.3.1 Plasmid as a vector</h5> <ul> <li>Plasmids are circular, double-stranded, extrachromosomal DNA capable of autonomous replication, occurring naturally in bacteria, archaea, and some eukaryotes.</li> <li>Plasmids can be integrated into chromosomes (episomes) and can contain genes providing benefits to the host cell, such as antibiotic resistance (R-plasmids) or toxin production (Col plasmids).</li> <li>Plasmids can be categorized based on copy number: high or multi copy plasmids (10-30 copies per cell) and low or single copy plasmids (1-few copies per cell).</li> <li>Development of plasmid-based vectors involves modifying, shortening, and recombining plasmids to incorporate desired properties, such as selectable markers and unique restriction sites.</li> <li>Further development includes the introduction of synthetic cloning sites (MCS) and easy selection markers, such as blue/white selection, facilitating the identification of recombinants. An example is the pUC19 vector.</li> </ul> <p>======</p> <h5 id="232-bacteriophage-as-vectors">2.3.2 Bacteriophage as vectors</h5> <ul> <li>Bacteriophage M13 is a filamentous bacteriophage of E. coli with a single stranded circular DNA genome of about 6.4 kb.</li> <li>The life cycle of M13 involves the replication of the single stranded M13 phage DNA inside the host to generate a double stranded replicative form (RF), which then changes to generate a single stranded genomic DNA (+) from double stranded RF.</li> <li>The circular single stranded genomic DNA (+) is assembled with capsid proteins during their passage through the host cell without causing cell lysis.</li> <li>The double stranded RF of the M13 genome is used for M13 vector construction, and the intergenic region is exploited for putting the insert so that it does not interfere with M13 replication.</li> <li>An example of M13 based vector for E. coli is M13mp18 which facilitates blue/white selection of the recombinants.</li> </ul> <p>======</p> <h5 id="233-cosmid-vector">2.3.3 Cosmid vector</h5> <ul> <li>A cosmid is a type of vector, combining features of plasmids and lambda phage.</li> <li>It has replication functions, unique restriction endonuclease sites, and selective markers from plasmid DNA.</li> <li>A lambda DNA segment, including the joined cohesive ends (cos sites), enables packaging in vitro into lambda phage coats.</li> <li>The minimal length of lambda DNA required for cos sites is 250 bp, which contains sequences for binding to and cleavage by the terminase enzyme.</li> <li>Cosmid cloning vectors can accommodate DNA inserts up to 45 kbp.</li> </ul> <p>======</p> <h5 id="234-phasmids-phagemids">2.3.4 Phasmids (Phagemids)</h5> <ul> <li>Phasmids are hybrid vectors that combine features of phage and plasmid.</li> <li>They are linear duplex DNAs with lambda phage DNA at the ends, containing genes for lytic infection, and a linearized plasmid in the middle.</li> <li>Both lambda phage and plasmid replication functions are intact in phasmids.</li> <li>Phasmid recombinants are packaged in vitro and can infect E. coli cells, replicating like a phage and forming plaques.</li> <li>If the vector contains the gene for the lambda repressor, the phasmid replicates as a plasmid instead of a phage.</li> </ul> <p>======</p> <h5 id="24-eukaryotic-host-vector-system">2.4 Eukaryotic Host Vector System</h5> <ul> <li>Plasmids are DNA molecules that can exist independently of chromosomal DNA, with a size of less than 10 kb.</li> <li>Bacteriophages, also known as phages, are viruses that infect bacteria, having a size range of 8-25 kb.</li> <li>Cosmids are plasmids that have been modified to include a bacterial origin of replication and a selectable marker, with a size ranging between 23-40 kb.</li> <li>PACs (P1-derived artificial chromosomes) and BACs (Bacterial artificial chromosomes) are vectors used in cloning large DNA fragments, with sizes up to 300 kb for BACs and between 100-300 kb for PACs.</li> <li>YACs (Yeast artificial chromosomes) are vectors used in cloning very large DNA fragments, with a size range of 200-500 kb.</li> </ul> <p>======</p> <h5 id="25-expression-vectors">2.5 Expression Vectors</h5> <ul> <li>Expression vectors are used to propagate DNA and allow efficient expression of the DNA insert (gene).</li> <li>These vectors can be plasmids or viruses based and contain an efficient promoter for expression of the cloned genes, often inducible for regulation.</li> <li>Unique restriction sites and a proper transcription termination sequence are provided for insertion and correct termination of the cloned gene.</li> <li>The cloned gene is inserted between a promoter and a terminator in an expression vector, also known as an expression cassette or sandwich expression vector.</li> <li>Heterologous gene expression, or expression of a gene from a different source than the host, requires the gene to not contain introns and not require post-translational modification for biological activity.</li> </ul> <p>======</p> <h5 id="26-shuttle-vectors">2.6 Shuttle Vectors</h5> <ul> <li>Shuttle vectors are developed for replication in two alternative hosts, either prokaryotic or eukaryotic.</li> <li>They are constructed by recombinant DNA techniques and many different types exist.</li> <li>Shuttle vectors can transfer between two prokaryotic species, or between a prokaryote (usually E. coli) and eukaryotic cells (including yeast, plants, and animals).</li> <li>Most eukaryotic vectors are shuttles, containing two origins of replication.</li> <li>In a given host, only one origin is active at a time.</li> </ul> <p>(Note: The figure showing the shuttle vector for E. coli and yeast has been omitted as per the instructions.)</p> <p>======</p>