Types of cloning Vectors for gene transfer
- 1. Vectors • Cloning vectors are DNA molecules that are used to "transport“ DNA sequences between biological hosts and the test tube. • Most vectors are genetically engineered. • A vector is used to amplify a single molecule of DNA into many copies.
- 2. Plasmids: Varieties Plasmids: Varieties Relaxed plasmids or high copy number plasmids Stringent plasmids or low copy number plasmids o Narrow-host range plasmids o Wide host range plasmids Naturally occurring plasmids Synthetic plasmids. Based on copy numbers Based on host range Based on occurence
- 3. Col plasmids: Col plasmids contain genes that make bacteriocins (aka colicins), which are proteins that kill other bacteria and thus defend the host bacterium. Resistance (R) plasmids: Resistance or R plasmids transfer through conjugation and contain genes that help a bacterial cell defend against environmental factors such as poisons or antibiotics. Fertility (F) plasmids: Fertility plasmids, also known as F-plasmids fall under the broad category of conjugative plasmids and contain transfer genes that allow genes to be transferred from one bacteria to another through conjugation. Virulence (Vir) plasmids: A virulence plasmid turns a host bacterium into a pathogen, which is an agent of disease. Degradative plasmids: These help the host bacterium to digest compounds that are not commonly found in nature, such as camphor, xylene, toluene, and salicylic acid Based on specific function/role
- 4. Bacteriophage Bacteriophages are a group of viruses that used bacterial cells as host and reproduce by infecting bacterial cells. Like all other viruses bacteriophages are also consisting of a protein coat that provides protection to the genome. Most of the phages are DNA viruses that code for several genes responsible for virus replication.
- 5. Types of phage vectors Types of phage vectors 20-25kb insert capacity
- 6. Cosmids Cosmids Plasmid derived vector - containing cos sites from phage are known as cosmids. Reported by Collins and Hohn, 1978. Cosmids can be used to clone large fragments of DNA by exploiting the phage in vitro packaging system.
- 7. Phasmids (Phagemids) • A phagemid or phasmid is a DNA-based cloning vector, which has both bacteriophage and plasmid properties. • They carry an origin of replication derived from bacteriophage (f1) in addition to origin of plasmid replication. • Phagemid vectors differ by having the ability to be packaged into the capsid of a bacteriophage, due to their having a genetic sequence that signals for packaging. • Phagemids are used in a molecular biology technique called "Phage Display“ (to study protein-protein interactions and other ligand/receptor combinations.)
- 8. Shuttle vectors • A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. • DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. • Main advantage of these vectors is they can be manipulated in E. coli, then used in a system which is more difficult or slower to use (e.g. yeast). • Shuttle vectors can propagate in eukaryotes and prokaryotes (e.g. both Saccharomyces cerevisiae and Escherichia coli) or in different species of bacteria (both E. coli and Rhodococcus erythropolis). There are also adenovirus shuttle vectors, which can propagate in E. coli and mammals.
- 9. Yeast Artificial Chromosome (YAC) is a vector used to clone DNA fragments larger than 100 kb and up to 3000 kb. First described in 1983 by Murray and Szostak, a YAC is an artificially constructed chromosome that contains a centromere, telomeres and an autonomous replicating sequence (ARS) element, which are required for replication and preservation of YAC in yeast cells. YACs are useful for the physical mapping of complex genomes and for the cloning of large genes.
- 10. PAC – P1 derived Artificial Chromosome PAC, is a DNA construct derived from the DNA of P1 bacteriophages and BAC. Efficient cloning vector to clone DNA fragments (100- to 300-kb insert size; average,150 kb) in E coli cells. P1 was developed as a cloning vector by Nat Sternberg and colleagues in the 1990s
- 11. Applications of PAC building genome libraries for human, mouse, etc, helps with projects such as Human Genome Project libraries served as the template for gene sequencing genome analysis on specific functions of different genes for more complex organisms (plants, animals etc.) facilitate gene expression