Host–Vector System

The host–vector system is essential in rDNA technology because gene cloning works only when the insert DNA is carried by a suitable vector into a compatible host. The vector supplies the DNA sequences needed for replication in the host, while the host supplies the enzymes and proteins required for copying the recombinant DNA. After introduction, the recombinant DNA is propagated and may also be expressed using the host’s genetic machinery. So, gene cloning is basically a two-component system of host + vector.

A good vector must have these major characteristics:

1. It should be small in size so it can be easily introduced into the host and should accommodate a fairly large insert.
2. It must have an origin of replication (ori) so it can replicate autonomously inside the host.
3. It should contain unique restriction sites so the foreign DNA can be inserted at a specific place.
4. It must have at least one selectable marker to screen transformants, such as **amp$^R$ or tet$^R$**.

These features make a DNA molecule useful for cloning.

A plasmid is a circular, double-stranded, extra-chromosomal DNA molecule capable of autonomous replication inside a host cell. It is inherited by daughter cells during cell division.

Types of plasmids mentioned in the chapter:
- Episomes: plasmids that can integrate into the host chromosome.
- R-plasmids: carry genes for antibiotic resistance.
- Col plasmids: produce toxins called colicins.
- F-plasmids: carry transfer genes for formation of pilus and transfer to other cells.
- Based on copy number:
- High or multi-copy plasmids: many copies per cell; show relaxed replication.
- Low or single-copy plasmids: one or few copies per cell; show stringent replication.

The plasmid cloning vector pBR322 was developed by modifying a natural plasmid to make it more useful for cloning.

Strategy used:
- The original plasmid pBR313 had useful features like relaxed replication, two selectable markers (**tet$^R$ and amp$^R$), and unique restriction sites.
- But it was too large, about 9 kb.
- Since more than half of its DNA was not essential, the non-essential part was deleted.
- This produced pBR322, whose size was reduced to 4,361 bp.
- pBR322 retained the ori, the selectable marker genes, and the unique cloning sites, making it a widely used cloning vector.

Thus, pBR322 is a shortened and reconstructed plasmid vector** designed for efficient gene cloning.

Lambda (λ) bacteriophage is a virus that infects E. coli. It has a head (capsid) and a tail. The head contains the phage genome, and the tail helps inject the genome into the bacterial cell.

Structure and life cycle:
- The λ genome is linear double-stranded DNA of 48,490 bp.
- Each end has 12-base single-stranded cohesive ends (cos sequences).
- Inside the phage particle, the genome remains linear.
- After entering E. coli, the cos ends pair and are ligated to form a circular DNA molecule.
- It can follow either the lytic cycle or the lysogenic cycle.
- In lysogeny, the phage DNA integrates into the host chromosome as a prophage.

Role of lambda phage-based vectors:
- λ vectors are widely used for gene cloning in E. coli.
- They are especially useful for larger DNA inserts than plasmids.
- The middle non-essential region of the λ genome can be replaced by foreign DNA.
- Recombinant DNA must be about 38–52 kbp for proper packaging into the λ head.
- Common λ vectors include λgt10, λgt11, and λEMBL3.
- They are used as insertion vectors or replacement vectors.

So, λ phage vectors are efficient cloning vectors for large DNA fragments.

M13 is a filamentous bacteriophage of E. coli. Its genome is a single-stranded circular DNA of about 6.4 kb, packed in a tube-like capsid.

Key features of M13 vectors:
- M13 infects only E. coli cells harbouring the F plasmid.
- Attachment occurs through the pilus.
- Inside the host, the single-stranded DNA forms a double-stranded replicative form (RF).
- Later, single-stranded genomic DNA is produced and released without causing cell lysis.
- For vector construction, the double-stranded RF DNA is used.
- The intergenic region is used for insertion of foreign DNA so that replication is not disturbed.
- M13 based vectors can clone large DNA, more than 42 kb.
- Example: M13mp18, which allows blue/white selection.

Application:
- Used for gene cloning in E. coli.
- Useful for blue/white screening and for obtaining single-stranded DNA.

Cosmids and phagemids are both hybrid vectors, but they differ in structure and use.

### Cosmids
- A cosmid is a hybrid vector that replicates like a plasmid but can be packaged in vitro into λ phage coats.
- It has plasmid features such as ori, selectable marker, and restriction sites, along with λ cos sites.
- It can carry large inserts up to about 45 kbp.

### Phagemids
- Phagemids are true hybrid vectors between phage and plasmid.
- They are linear duplex DNAs with λ phage DNA ends and a linearised plasmid middle region.
- Both phage and plasmid replication functions are intact.
- They are packaged in vitro before infection.
- In an E. coli cell, they can replicate like a phage and form plaques.
- If they contain the λ repressor gene, they replicate as a plasmid rather than as a phage.

### Difference in one line
- Cosmid: plasmid-like vector with λ cos sites; mainly used for large inserts.
- Phagemid: phage-plasmid hybrid with λ ends and plasmid middle; can behave either like a phage or a plasmid depending on the construct.