Biotechnology: Class 12 — Chapter 2: Host–Vector System

1. Insert Isolation: The target gene or desired DNA molecule (termed the insert) is isolated from the source organism.
2. Vector Ligation: The target gene is inserted into a convenient carrier DNA molecule called a vector, producing recombinant DNA (rDNA).
3. Host Transformation: The rDNA is introduced into a living organism, referred to as the host, where it undergoes propagation and expression under the term gene cloning.

• Escherichia coli: The most extensively used prokaryotic host; a rod-shaped, Gram-negative bacterium found in the lower intestine of warm-blooded organisms. It grows rapidly, doubling its population approximately every 20 minutes. The K12 strain is the most commonly used in gene cloning.
• Bacillus subtilis: An important alternative prokaryotic host, particularly utilized when the experimental goal is the active secretion of the protein encoded by the cloned gene.

• Saccharomyces cerevisiae: Baker's yeast; the most widely used eukaryotic host. It multiplies asexually by budding, grows as single cells in suspension, and has a long history of safe use in the food industry, classifying it as a Generally Recognised As Safe (GRAS) organism.

1. Small Size: Facilitates easy incorporation into the host cell and allows integration of larger inserts.
2. Origin of Replication (ori): Required to enable autonomous replication inside the host organism.
3. Unique Restriction Sites: Necessary for precise cleavage; multiple sites for a single enzyme would fragment the vector.
4. Selectable Markers: Genes required to screen and identify transformants, such as antibiotic resistance genes like tetracycline resistance ($tet^R$) and ampicillin resistance ($amp^R$).

Plasmids are circular, double-stranded (ds), extra-chromosomal DNA molecules capable of autonomous replication, ranging in size from a few thousand base pairs to over 100 kilobase pairs (kbp).

• Episomes: Plasmids that can integrate directly into the host cell's chromosomes.
• R-plasmids: Drug-resistance plasmids encoding enzymes that deactivate antibiotics (e.g., ampicillin, tetracycline, chloramphenicol).
• Col plasmids: Plasmids that produce toxins called 'colicins' to kill competing bacteria.
• F-plasmids: Fertility plasmids containing 'transfer genes' encoding a pilus for conjugative transfer between bacterial cells.

• High or Multi-Copy Plasmids: Replication is independent of host chromosomal DNA control, yielding 10–30 copies per cell via relaxed replication. This provides higher yields and stronger signals during screening.
• Low or Single-Copy Plasmids: Replication is strictly controlled alongside host chromosomal DNA, limiting the plasmid to one or a few copies per cell via stringent replication.

• pBR313: An early plasmid vector replicating in a relaxed fashion with $tet^R$ and $amp^R$ markers, but limited by its large size (9 kb).
• pBR322: Constructed by deleting non-essential sequences of pBR313, reducing the size to 4,361 bp. It contains unique cloning sites for restriction enzymes (PstI, EcoRI, BamHI, SalI, PvuII) and $amp^R$/$tet^R$ markers.
• Multiple Cloning Site (MCS): Also known as a polylinker; a short synthetic DNA fragment containing many unique restriction sites to facilitate foreign DNA insertion.
• pUC19: An improved 2.686 kb cloning vector containing an MCS, $amp^R$, and a portion of the $\beta$-galactosidase (lacZ) gene of *E. coli* to enable blue/white selection of recombinants.

A virus infecting *E. coli* consisting of a head (capsid) containing a 48,490 bp linear double-stranded DNA genome, and a tail.

• Lytic Cycle: The phage DNA replicates, produces new phage particles, and lyses the host cell to release progeny.
• Lysogenic Cycle: The phage DNA integrates into the host chromosome as a prophage, replicating silently alongside host division.
• cos Sequences: 12-base single-stranded complementary cohesive ends at each terminus of the linear genome. Inside the host, they pair and ligate to form a circular genome with a cos site (48,502 bp).
• Rolling Circle Replication: Generates long concatemers of phage genomes joined end-to-end, which are cleaved at *cos* sites during packaging into phage heads.
• Vector Engineering: Approximately one-third of the central genome (the stuffer region) is non-essential for lytic infection and can be replaced with insert DNA. Recombinant DNA must be 38 to 52 kbp for packaging.
• Insertion Vectors: Have a single restriction site for insert DNA (e.g., $\lambda$gt10, $\lambda$gt11).
• Replacement Vectors: Have a pair of cloning sites flanking a removable non-essential stuffer region (e.g., $\lambda$ EMBL3).

A filamentous bacteriophage of *E. coli* containing a 6.4 kb single-stranded circular DNA genome packaged in a tubular capsid. It infects only *E. coli* cells harboring an F plasmid via the pilus.

• Replication Cycle: The single-stranded (+) DNA replicates to form a double-stranded replicative form (RF). The RF is used for vector construction, and the mode of replication later switches to generate single-stranded (+) genomic DNA for packaging without cell lysis.
• M13mp18: A common M13-based vector containing an MCS within a *lacZ* gene fragment to facilitate blue/white selection.

• Cosmid Vector: A hybrid vector containing plasmid replication functions, unique restriction sites, and selective markers, combined with a lambda phage *cos* site. It is packaged *in vitro* into phage coats using the enzyme terminase and can accommodate inserts up to 45 kbp.
• Phasmids (Phagemids): Hybrid vectors containing lambda phage ends (with lytic genes) and a linearized plasmid middle. They can replicate as a phage or as a plasmid (if they encode the lambda repressor CI).
• Yeast Artificial Chromosomes (YACs): Designed for cloning very large DNA fragments (200–500 kb). They contain two yeast telomeric sequences (Tel), a centromeric sequence (Cen), an autonomously replicating sequence (ARS), and selectable markers (e.g., TRP1, URA3). YACs exist in a circular form for bacterial propagation and a linear form for yeast propagation.
• Bacterial Artificial Chromosomes (BACs): High-capacity cloning vectors derived from bacterial plasmids, accommodating inserts up to 300 kb.
• Phage Artificial Chromosomes (PACs): High-capacity vectors derived from bacteriophage components, accommodating inserts of 100–300 kb.

Expression vectors act as vehicles for DNA propagation and contain regulatory sequences to allow the cloned gene to be expressed efficiently.

• Expression Cassette: A transcription unit consisting of an inducible promoter (5' end), an operator, a ribosome binding site (RBS), unique cloning sites, and a transcription termination sequence (3' end). Also known as sandwich expression vectors.
• Heterologous Gene Expression: The expression of a foreign gene in a host of a different species (e.g., eukaryotic gene in a prokaryotic host).

Eukaryotic genes often fail to express functionally in prokaryotic hosts due to structural differences in transcription and translation machinery.

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Engineering Requirements for Heterologous Expression: (i) The eukaryotic gene must lack introns because prokaryotes lack splicing machinery; (ii) The expressed protein must not require post-translational modifications (such as glycosylation) for its biological activity.

Shuttle vectors are designed to replicate in two alternative host species (e.g., between *E. coli* and yeast, plants, or animal cells).

• Dual Origins: They contain two distinct origins of replication, though only one origin is active at any given time depending on the host cell environment.