Cloning Vectors: Plasmid and Cosmid Characteristics

Plasmid as a Cloning Vector

1. Circular double-stranded DNA capable of independent replication: Plasmids replicate autonomously inside bacteria using their own origin of replication, ensuring multiple copies.
2. Presence of origin of replication (ori): The ori controls copy number and guarantees that inserted genes duplicate with the host cell.
3. Selectable marker genes for screening: Antibiotic-resistance genes (e.g., ampicillin) allow only transformed cells to survive, simplifying identification.
4. Multiple cloning site (MCS) for easy insertion: Contains clustered restriction sites enabling precise cutting and insertion of foreign DNA.
5. Small size enhances transformation efficiency: Smaller plasmids enter host cells more easily and are maintained stably.
6. Low copy vs high copy options: Researchers choose copy control depending on protein expression needs and plasmid stability.
7. Non-integrative and safer: Plasmids usually remain episomal and do not disrupt host chromosomes.
8. Reporter genes enable blue-white screening: Insertional inactivation of lacZ distinguishes recombinants visually.
9. Compatibility with many host species: Engineered plasmids work in E. coli and other bacteria, and some shuttle vectors work across species.
10. Easy isolation and manipulation in vitro: Plasmids are simple to purify, cut, ligate, and re-introduce into hosts repeatedly.
11. Example: pBR322 and pUC19 are classical plasmid vectors widely used for cloning and expression.

Flow: Cut plasmid → Insert foreign DNA → Transform host → Select recombinants

Cosmid as a Cloning Vector

1. Hybrid between plasmid and bacteriophage $\lambda$ DNA: Cosmids combine plasmid features with phage cos sites to carry larger inserts.
2. Contain cos sites for packaging: Cos sequences permit in vitro packaging into $\lambda$ phage heads for high-efficiency delivery.
3. Large cloning capacity: Cosmids can accommodate ~35–45 kb DNA, much larger than most plasmids.
4. Selectable markers and ori retained: Antibiotic markers and replication origins allow maintenance like plasmids after entry.
5. No phage genes for lytic cycle: Once inside, cosmids replicate as plasmids without producing phage particles, improving stability.
6. Reduced rearrangements due to fewer repeats: Simplified structure lowers the risk of insert instability.
7. Useful for genomic library construction: Ideal for cloning long genomic fragments retaining gene context.
8. Requires packaging step before infection: DNA is packaged into phage particles then introduced into bacteria, boosting efficiency.
9. Screening similar to plasmids: Antibiotic selection and additional markers identify positive clones.
10. Bridges gap between plasmids and BAC/YAC systems: Offers mid-range capacity without complex handling.
11. Example: Cosmids derived from pJB8 enabled construction of early eukaryotic genomic libraries.

Flow: Ligate insert → Package with cos sites → Infect bacteria → Select stable clones