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Plasmid and Bacteriophage λ as Cloning VectorsBY: Nidhi Uppangala | Category: Others | Submitted: 2010-09-01 16:22:30
Many types or kinds of cloning vectors are used for the transfer of gene of interest into the desired tissue or cell. In other words vectors are used as vehicle to carry gene of interest. Several types of vectors are being constructed in laboratory; each vector has got different molecular properties as well as cloning capacity. Some of the examples for vectors used in recombinant DNA technology are plasmids, phagemids, cosmids, shuttle vectors and much more.
Plasmid as Cloning Vector:
1. Plasmids are extra chromosomal circular double stranded DNA, present in bacterial cells. Plasmid are used as cloning vectors, include ori site or origin of replication, needed for replication in the bacterial cells. For an example E.coli plasmid vector, should have following features:
a. Ori site for replication.
b. Selectable marker genes, such as antibiotic resistance gene
c. Unique restriction sites, so that the restriction enzymes can be used to cut the plasmid and DNA of interest can be inserted into the plasmid.
2. pUC19 is an example for an E.coli cloning vector. pUC19 is basically 2,686bp in length. The features of this pUC19 plasmids are as follows:
a. High copy number, as high as nearly hundred copies per bacterial cell. This helps in getting good yield of cloned DNA in short duration.
b. It also has got selectable marker as ampicillin resistance gene.
c. This plasmid also got a cluster of unique restriction sites known as polylinkers or in other words this plasmid has got multiple cloning sites.
d. The polylinkers or multiple cloning sites are also a part of lacZ (galactosidase) gene. That is pUC19 plasmid will complement a β lacZ E.coli allowing it to become lacZ positive. When DNA of interest is cloned into the polylinkers lacZ is disrupted, this prevents the complementation from occurring.
e. A chromogenic analog of lactose known as X-gal, turns blue in presence of galactosidase, and remains white in β absence; therefore blue white colonies can be used as screening method to identify the bacterial colonies which contain recombinant plasmid.
3. DNA of interest is inserted into a cloning vector using restriction enzyme and then ligating it with enzyme ligase.
a. pUC19 plasmid vector can be cut using restriction enzyme that has got a unique site in the polylinker region.
b. DNA of interest is also cut using the same restriction enzyme.
c. DNA of interest and pUC19 DNA are mixed and allowed for random joining of fragments.
d. Resulting plasmids then transformed into E.coli cells either by chemical treatment method or by electroporation method. Then these bacterial cells are grown on media contain ampicillin and Xgal.
e. Ampicillin resistance is resulted from pUC19 sequence, Blue colonies are resulted from the rejoined plasmids, and white colonies will result from transformed bacterial cells with recombinant plasmids.
4. Many different types of cloning vectors are designed in the laboratory and they are commercially available for use. These vectors have different array of unique restriction sites in the polylinker site.
5. Many plasmid cloning vectors are designed and available for many prokaryotic organisms as well as for eukaryotic organisms.
6. But the plasmid cloning vectors which carry more than 5 to 10 kb gene of interest are often unstable. Therefore size of the gene of interest is limited in plasmid cloning vectors.
Bacteriophage λ as Cloning Vector:
1. There are many versions or types of bacteriopage λ used as cloning vectors. For example sequence of the gene responsible for the lysogeny is removed, so that only one type that is lytic infection is possible in this bacteriophage λ vector.
2. The central region of the λ chromosome is used for inserting the gene of interest. Gene of interest is inserted using restriction digestion and also DNA ligation method.
3. When this ligated DNA is mixed with phage proteins, this leads to the assembling of phage head and DNA is also packed to form virus particles.
4. Only viral particles with both the phage chromosome arms and also with proper central segment (3752kb) are able to replicate by infecting the E.coli bacterial cell. Progeny phages contain DNA of interest; hence provide a large quantity of DNA of interest.
5. Many types of phage cloning vectors are available with varying features like an expanded array of restriction sites and much more.
Plasmids and bacteriophage λ are most commonly used cloning vectors in biotechnology applications or in particular in restriction digestion technology.
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