Host–Vector System Chapter Notes | Biotechnology for Class 12 - NEET PDF Download

Two Key Components of Recombinant DNA Technology

As discussed in the previous chapter (Chapter 1), rDNA technology refers to joining two different DNA molecules with an aim to isolate, propagate, characterise and manipulate the genes for various applications. This technique involves two major steps (Fig. 2.1). In the ￿￿rst step, the desired DNA molecule, generally termed as insert (target gene), is isolated from the source. In the second step, this target gene is inserted into a convenient carrier DNA molecule called vector. The vector containing the insert is called recombinant DNA (rDNA). Subsequently, the rDNA is introduced into an organism referred to as host. Using genetic machinery of the host, the rDNA undergoes propagation and expression. This whole process of rDNA technology is covered under the term ‘gene cloning’. Thus, gene cloning may be considered as a two component system: a compatible host and a vector, where the vector provides essential sequences required for its replication in a compatible host, which provides various replication functions (enzymes and proteins).

Host

A large number of host organisms, both prokaryotic and eukaryotic are used for gene cloning (Fig. 2.2). A host should allow easy entry of the rDNA into the cell and should not consider the recombinant DNA as a foreign DNA and degrade it. The host must supply all the required enzymes and proteins for smooth replication of the vector DNA along with insert. A wide variety of genetically de￿￿ned strains are available as hosts. Among prokaryotic hosts, E. coli is the most extensively used. Typical E. coli is a rod-shaped Gram-negative bacterium commonly found in the lower intestine of warmblooded organisms. It is able to reproduce and grow rapidly, doubling its population about every 20 mins. K12 strain of E. coli is one of the most commonly used hosts in gene cloning. Other prokaryotic hosts have also been developed. For example, Bacillus subtilis constitutes an important alternative host, where the aim is secretion of a protein encoded by a cloned gene. Among eukaryotic hosts, the most widely used is yeast.

Vector

• A vector is any DNA molecule capable of self-replication inside a host cell, serving as a vehicle for gene cloning.
• An ideal vector should be small in size to facilitate easy incorporation into the host cell while being able to accommodate a large DNA insert.
• Vectors must possess an origin of replication (ori) to enable autonomous replication within the host organism.
• Vectors require unique restriction sites to allow precise insertion of foreign DNA without fragmenting the vector into multiple pieces.
• At least one selectable marker, such as antibiotic resistance genes (e.g., tetracycline resistance, tet^R, or ampicillin resistance, amp^R), is necessary to screen for transformants.
• Vectors can be prokaryotic or eukaryotic, with examples including plasmids, bacteriophages, cosmids, phasmids, and artificial chromosomes (Fig. 2.2).

Plasmid as a Vector

• Plasmids are circular, double-stranded, extra-chromosomal DNA molecules capable of autonomous replication in bacteria, archaea, and some eukaryotes like yeast.
• Plasmid sizes range from a few thousand base pairs to over 100 kilobase pairs (kbp).
• Like host chromosomal DNA, plasmid DNA is replicated before cell division, ensuring at least one copy is segregated to each daughter cell.
• Some plasmids, known as episomes, can integrate into the host chromosome.
• Naturally occurring plasmids often carry beneficial genes, such as those encoding antibiotic resistance (R-plasmids), toxins (Col plasmids), or transfer genes for conjugation (F-plasmids).
• R-plasmids encode enzymes that deactivate antibiotics like ampicillin, tetracycline, and chloramphenicol, making them valuable for gene cloning.
• Col plasmids produce colicins, toxins that kill other bacteria, enhancing host survival.
• F-plasmids encode proteins forming a pilus, enabling plasmid transfer to other bacterial cells of the same or related species.
• Plasmids are classified 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).
• High-copy plasmids undergo relaxed replication, independent of bacterial chromosomal DNA replication control, yielding higher recombinant DNA quantities.
• Low-copy plasmids undergo stringent replication, synchronized with bacterial chromosomal DNA replication, limiting their copy number.
• Plasmid vectors are developed by modifying naturally occurring plasmids through in vivo and in vitro recombination to incorporate desired properties, such as unique restriction sites and selectable markers.
• The plasmid pBR313, with relaxed replication, high copy number, and selectable markers (tet^R and amp^R), was initially used but was large (9 kb).
• Non-essential sequences of pBR313 were deleted to create pBR322, a smaller vector (4,361 bp) with an origin of replication, unique restriction sites (e.g., PstI, EcoRI, BamHI, SalI, PvuII), and antibiotic resistance genes (amp^R, tet^R), making it widely used for gene cloning.
• Further advancements introduced multiple cloning sites (MCS) or polylinkers, short synthetic DNA fragments with numerous unique restriction sites for flexible DNA insertion.
• Improved selection methods, such as blue/white selection using the lacZ gene (encoding β-galactosidase), were incorporated into vectors like pUC19, which includes an ori, amp^R, and part of lacZ for recombinant screening.

Bacteriophage as Vectors

• Bacteriophage vectors, derived from bacteriophage genomes (viruses that infect bacteria), are more efficient than plasmids for cloning large DNA inserts and screening large numbers of recombinants.
• Screening bacteriophage plaques (lysed bacterial cells due to phage infection) is more efficient than screening bacterial colonies.
• The most common bacteriophages used for vector construction are lambda (λ) and M13, both designed for E. coli hosts.

Lambda (λ) Phage Vector

• The lambda bacteriophage, a virus infecting E. coli, is widely used as a cloning vector due to its ease of propagation and well-studied biology.
• The λ phage consists of a head (capsid) containing the genome and a tail for injecting DNA into the host cell.
• The λ phage has two life cycles: lytic, where the phage DNA replicates, produces new phage particles, and lyses the host cell, and lysogenic, where the phage DNA integrates into the host chromosome as a prophage without harming the host.
• The λ genome is linear double-stranded DNA (48,490 bp) with 12-base single-stranded cohesive ends (cos sequences) that are complementary, enabling circularization inside the host cell.
• Upon entering the E. coli cell, the cos sequences pair, and host enzymes ligate them to form a circular genome (48,502 bp) with a sealed cos site.
• The λ genome includes an origin of replication, genes for head and tail proteins, and enzymes for lytic and lysogenic cycles.
• During lytic multiplication, the λ genome undergoes rolling circle replication, producing a concatemer of multiple genome copies joined by cos sites, which are cleaved to package single genomes into phage heads.
• Approximately one-third of the λ genome (the middle region, containing lysogeny genes) is non-essential for lytic infection and can be replaced with foreign DNA inserts.
• Recombinant λ DNA must be 38–52 kbp for efficient packaging into the phage head.
• Lambda vectors are classified as insertion vectors (with a single restriction site for DNA insertion) or replacement vectors (with two restriction sites flanking a dispensable stuffer region replaced by the insert).
• Common λ-based vectors include λgt10 (insertion vector, 6 kbp insert, lytic selection), λgt11 (insertion vector, 7.2 kbp insert, blue/white selection), and λEMBL3 (replacement vector, 20 kbp insert, lytic selection).

Bacteriophage M13

• M13 is a filamentous bacteriophage of E. coli with a single-stranded circular DNA genome (6.4 kb) packaged in a tube-like capsid.
• The M13 genome encodes genes (I–X) for coat proteins, replication, and assembly, with a small non-essential intergenic region flanking the origin of replication.
• M13 infects E. coli cells harboring an F-plasmid, which express a pilus for phage attachment and adsorption.
• Upon infection, the single-stranded M13 DNA is converted into a double-stranded replicative form (RF) inside the host.
• Replication later shifts to produce single-stranded genomic DNA (+) from the RF, which is packaged into new phage particles without lysing the host cell.
• The double-stranded RF DNA is used for vector construction, with inserts placed in the intergenic region to avoid disrupting replication.
• M13 vectors can clone large DNA inserts (up to 42 kb).
• An example is M13mp18, which facilitates blue/white selection of recombinants using the lacZ gene.

Cosmid Vector

• Cosmids are hybrid vectors combining plasmid and lambda phage properties, replicating like plasmids but packageable into lambda phage coats in vitro.
• A typical cosmid includes plasmid-derived replication functions, unique restriction sites, selectable markers, and a λ DNA segment with cohesive ends (cos sites).
• Only 250 bp of λ DNA, including the cos junction and terminase-binding sequences, is needed for packaging.
• Cosmids can accommodate large DNA inserts, up to 45 kbp, making them suitable for cloning sizable genomic fragments.

Phasmids (Phagemids)

• Phasmids are hybrid vectors combining phage and plasmid features, consisting of linear duplex DNA with lambda phage DNA ends (containing lytic infection genes) and a linearized plasmid middle region.
• Phasmids retain both lambda phage and plasmid replication functions, allowing them to replicate as phages or plasmids depending on conditions.
• Phasmid recombinants are packaged in vitro before infecting E. coli, where they form plaques like phages.
• If the phasmid contains the lambda repressor gene, it replicates as a plasmid rather than a phage.

Eukaryotic Host Vector System

• Eukaryotic gene cloning requires vectors capable of handling large DNA fragments, as eukaryotic genes often contain introns spanning hundreds of kilobases.
• Special vectors, such as artificial chromosomes, have been developed to clone large eukaryotic DNA fragments.
• The baker’s yeast, Saccharomyces cerevisiae, is the most common eukaryotic host due to its genetic tractability and safety.
• S. cerevisiae reproduces asexually by budding, grows as single cells in suspension, and forms colonies on solid media, similar to E. coli.
• A large collection of genetically mapped metabolic, biosynthetic, and cell cycle mutants of S. cerevisiae is available for research.
• S. cerevisiae is classified as Generally Recognized As Safe (GRAS) due to its long history of safe use in the food industry.
• S. cerevisiae harbors a 2 µm circular plasmid, which has been used to develop cloning vectors.
• Yeast Artificial Chromosomes (YACs) are designed to clone large DNA inserts (200–500 kb).
• YACs contain two yeast telomeric sequences (chromosome ends), a yeast centromeric sequence, an autonomously replicating sequence (ARS) for replication initiation, and selectable markers.
• YACs exist in a circular form for propagation in bacteria (with a bacterial ori and antibiotic resistance gene) and a linear form for use in yeast, with telomere sequences at each end.

High Capacity Cloning Vectors

• High-capacity vectors, such as YACs, Bacterial Artificial Chromosomes (BACs), and Phage Artificial Chromosomes (PACs), are designed to clone large DNA fragments.
• Insert size capacities vary: plasmids (≤10 kb), bacteriophages (8–25 kb), cosmids (23–40 kb), PACs (100–300 kb), BACs (≤300 kb), and YACs (200–500 kb).

Expression Vectors

• Expression vectors are designed to propagate DNA inserts and enable efficient gene expression, unlike cloning vectors focused solely on propagation.
• Expression vectors can be plasmid- or virus-based and contain an inducible promoter to regulate cloned gene expression.
• Unique restriction sites downstream of the promoter allow insertion of the gene to be expressed.
• A transcription termination sequence near the 3’ end ensures proper transcription termination.
• The gene is inserted between the promoter (5’ end) and terminator (3’ end), forming an expression cassette, earning these vectors the name “sandwich expression vectors.”
• Heterologous gene expression (e.g., eukaryotic gene in a prokaryotic host) requires the gene to lack introns (due to absent splicing in prokaryotes) and not depend on post-translational modifications (e.g., glycosylation) for functionality.

Shuttle Vectors

• Shuttle vectors are designed to replicate in two different hosts, typically a prokaryote (e.g., E. coli) and a eukaryote (e.g., yeast, plants, or animals).
• Constructed using recombinant DNA techniques, shuttle vectors contain two origins of replication, with only one active in a given host.
• Many eukaryotic vectors are shuttle vectors, enabling manipulation in bacteria before transfer to eukaryotic hosts.