HYBRID PLASMID / PHAGE VECTORS
4. HYBRID PLASMID / PHAGE VECTORS
4.1. Cosmids :
A cosmid cloning system: The cosmid contains an E.coli origin of replication (ori) that allows the cosmid to be maintained as a plasmid in E. Coli. The two intact cos sites closely flanking a unique ScaI site; a unique BamHI site near, but outside, one of the cos sites; and a Tetr gene. The source DNA is cut with BamHI and fractionated by size to isolate molecules that are about 45 kb long.
The plasmid DNA is cut with ScaI and BamHI. The two DNA samples are mixed and treated with T4 DNA ligase. After ligation, some of the joined DNA molecules will have a 45-kb piece of DNA inserted into the BamHI site of the plasmid; when this happens, the two cos sequences are about 50 kb apart. These molecules are packaged into bacteriophage λ heads in vitro, and infective particles are formed after the addition of tail assemblies. Infective bacteriophage λ delivers a linearized DNA molecule with cos extensions into E. Coli. After entry into the host cell, the cos ends base pair and the DNA ligase of the host cell seals the nicks. The circular DNA molecule that is created in this way persists as a plasmid in the host cell. In this case, transformed cells can be identified because they are resistant to the antibiotic tetracycline.
Cosmid is isrid between a phage DNA molecule and a bacterial plasmid. A cosmid is basically a plasmid that contains a cos site. The cos site help in invitro packaging of cosmid into bacteriophage. It also has a selectable marker, such as ampicillin resistance gene, and a plasmid of origins of replication. Cosmid lacks phase genes do not produce phage. Thus the colonies are seen for cosmid vector are just like plasmid vector.
cos site recognised l genome during packing by phage particles
cosmid only replication of genome DNA not form new phage partical (because of gene absent)
Antibiotic restant. Insertion size of DNA ~ 40 - 50 kb.
Use in form genome library
eg. pJB8 (5.4 kb).
This vector has packaging and infection similar to phage but since it lacks phage gene it acts as a plasmid in E. coli cell. This is generally small in size (4 – 6 kb) and can carry foreign DNA up to 47 kb. This is an efficient and specific method for introducing a recombinant DNA and its cloning capacity is about two times greater than the best replacement vector.
First time developed F-plasmid based vector name as pFOS by Simmon co-worker in 1992. It is carry -cos site and F-plasmid ori.
Insert DNA size is 40 kb.
Fosmid are used in metagenome library
Fosmid contain elements
1. ori T Origen of transfer by stating conjugation time
2. Tra Transfer gene (F-pilus gene)
3. Is Insertion Element (copy the different location)
4.2. Phagemids :
1. It is Hybrid of plasmid and M-13.
2. Origin of replication (ori) of a plasmid.
3. Intergenic region (IG region) which contains the packaging signal for the phage particle and also has replication origin inside phage.
4. A gene encoding phage coat protein.
5. A selection marker.
6. Restriction enzyme recognition sites.
Phagemids are the hybrid vector of plasmid and M-13 bacteriophage. The origin of replication in phagemid is of M-13 bacteriophage. This allows single-stranded recombinant DNA formation. The lacZ gene of phagemid has MCS site. Thus allows blue-white screening of colonies. Phagemid is used as a cloning vector, the sequence in vectors and expression vectors. lZAP vector family belongs to phagemids.
4.3. 2 m plasmid: This is a eukaryotic plasmid and is generally formed in yeast. It is small in size i.e., 6 kb which makes it advantageous to use as a vector. In the yeast cell, this vector has a copy number between 100-200. the origin of this plasmid can be used for replication. The enzymes required for replication is either provided by the host or can be encoded by REP1 and REP2 genes carried by DNA.
The recombinants of this type of plasmid can be distinguished by growing cell auxotrophic host. LEU2 gene can be used as a selectable marker. This gene codes for b-isopropyl-malate dehydrogenase. Which is responsible for the conversion of pyruvic acid to leucine. The cells that lack leu-2 gene cannot grow in auxotrophic mutant media. Leucine amino acid has to be provided in the growth medium. Recombinants can grow in auxotrophic mutant cells and hence can be selected. This selection is possible as the transformed cells have a copy LEU2 gene. In a cloning experiment, the cells are grown on minimal media. Minimal media is a culture media in which no amino acid is added explicitly. Only the transformed cells are able to form colonies as they have a gene for amino acid synthesis.
4.4. Yeast Episomal Plasmids: Vectors derived from 2m plasmid are known as yeast episomal plasmids. This plasmid can contain an entire 2 m plasmid or may have only the origin of replication of 2m plasmid. For example YEP13. This yeast episomal plasmid works as a shuttle vector. It contains the origin of replication of 2m plasmid and selectable LEU2 gene. In addition to this, it also includes the entire sequence of PBR322 and therefore is suitable for both yeast and E. coli cells. YEPs (Yeast episomal plasmids) are based on the natural 2mm plasmid of yeast, thus it can replicate in two manners. It may either integrate into yeast chromosome or may replicate independently.
4.5. Yeast Integrative plasmid: It is consist of E.coli vector such as PBR322, PUC19, pBulescript. It has yeast selection maker such as URA-3, His-3, TRP1, leu-2. They lack any replication origin for yeast. i.e. it can't be replicated in yeast thus they can be propagated only through integration into the genome.
4.6. Yeast replicative plasmids: Yeast replicative plasmids carry ARS that enable E.coli vectors to replicate in yeast cells. Sometimes the plasmids contain the sequences around centromere. These plasmids act as mini chromosomes and are known as yeast centromere plasmids.
4.7. Yeast centromeric plasmid–(YCP)– are autonomously replicating vectors containing centromere sequence (CEN) and ARS. Not useful as expression vector but use as a cloning vector, however, the copy number is low (1-3 per cell).
- TOOL AND TECHNOLOGY
- HYBRID PLASMID / PHAGE VECTORS
- ARTIFICIAL CHROMOSOMES
- SHUTTLE VECTORS
- ENZYMES USED FOR RECOMBINANT DNA TECHNOLOGY
- DNA LIBRARY
- FLUROSCENT ACTIVATED CELL SORTER
- DNA MICROARRAY OR GENE CHIP OR BIO CHIP
- ANTIBODY GENERATION
- RADIOIMMUNOASSAY (RIA)
- ELISA OR ENZYME LINKED IMMUNOSORBANT ASSAY
- POLYMERASE CHAIN REACTION
- TYPE OF HYDROLYSIS PROBE
- X-RAY DIFFRACTION
- NMR (NUCLEAR MAGNETIC RESONANCE)
- CIRCULAR DICHROISM
- DNA SEQUENCING
- TRANSGENIC ANIMALS
- CRE–LOX P RECOMBINANT SYSTEM
- GENE THERAPY
- TRANSGENIC PLANTS
- PLANT TISSUE CULTURE (PTC)
- MICRO PROPAGATION
- ARTIFICIAL SEEDS
- PRACTICAL APPLICATIONS OF PLANT TISSUE CULTURE
- ANIMAL CELL CULTURE