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Table of Contents
Plasmid is an extrachromosomal, small, circular double-stranded DNA that is distinct from a cell’s chromosomal DNA. The plasmid behaves as a cloning vector, providing the replicative ability that enables the cloned gene to be propagated inside the host cell. Plasmids can replicate efficiently in bacterial hosts because each plasmid contains an origin of replication (ori) which is recognized by the DNA polymerases and other proteins that normally replicate the bacterium’s chromosomes. The host cell’s replicative machinery therefore propagates the plasmid along with any new genes that have been inserted into it.
• pBR322 plasmid is small contains just 4363 bp
• Has origin of replication
• Carries genes that code enzymes which enable the host bacterium to survive in the presence of two antibiotics: ampicillin (amp) and tetracycline (tet) resistant.
The figure shows the position of origin of replication(ori), ampicillin resistance gene (ampR), tetracycline resistance gene (tetR) and recognition sequences for seven restriction endonucleases.
Mechanism of gene cloning with plasmid vector pBR322:
- An animal gene has been obtained from an animal DNA and a small E.coli plasmid has been purified and treated with the same RE, that cuts the plasmid in a single position.
- Then these two DNA molecules are added together with DNA ligase. So the injection of animal gene into the plasmid is completed.
- The recombinant plasmid is now reintroduced into E. coli, and the inserted gene does not disrupt its replicative ability. The plasmid is recognized by the DNA polymerases and other proteins that normally replicate the bacterium’s chromosomes.
- Then the plasmid as well as the inserted gene will be replicated and after the cell division, the daughter cells will possess the recombinant plasmid.
- More rounds of plasmid replication and cell division will cause in a colony of recombinant E. coli bacteria, each of which will contain multiple copies of the animal gene.
Screening of recombinant DNA using pBR322 vector:
After transformation, cells are plated onto ampicillin agar. All cells that contain a pBR322 plasmid, either recombinant or not, will divide and produce a colony. The colonies are then transferred onto tetracycline agar medium by replica plating, which results in the colonies on the second plate retaining the relative positions that they had on the first plate. Some colonies do not grow on the tetracycline agar because their cells contain recombinant pBR322 molecules that have a disrupted tetracycline-resistance gene. These are the colonies that we are looking for because they contain the cloned gene, so we go back to the ampicillin plate, from which samples of the cells can be recovered.
Replica plating is not a difficult technique, but it costs time. If we can distinguish recombinants from non-recombinants simply by plating onto a single agar medium it would be much better and time saving. This becomes possible with most of the modern plasmid cloning vectors, including pUC8.
pUC8 vector carries the ampicillin-resistance gene from pBR322, along with a second gene, called lacZ, which is a part of the E. coli gene for the enzyme β-galactosidase. The presence of functional β-galactosidase molecules in the cells can be checked by a histochemical test with a compound called X-gal in which the enzyme converts into a blue product. The lacZ gene contains a cluster of unique restriction sites that allow the insertion of desired DNA into any one of these sites that results in insertional inactivation of the gene and hence loss of β-galactosidase activity. Recombinants and non-recombinants can therefore be identified easily by plating the transformed cells onto agar medium containing ampicillin and X-gal. All colonies that grow on this medium are made up of transformed cells because only the transformants are ampicillin resistant. There we will see that some colonies are blue, and some are white. The colonies that are blue contain cells with functional β-galactosidase enzymes and hence the lacZ gene is undisrupted; these colonies are therefore non-recombinants. The white colonies comprise cells without β-galactosidase activity and hence with disrupted lacZ genes; these are the recombinants.
Advantages of using Plasmids (other than cosmid) as vectors:
- The small size makes it easy to manipulate and isolate.
- More stable because of circular configuration.
- Replicate independent of the host.
- High copy number.
- Detection is easy because of antibiotic-resistant genes.
Disadvantages of using Plasmids as vectors:
- Large fragments cannot be cloned.
- Size range is only from 0 to 10kb.
- Standard methods of transformation are inefficient.
- Cosmid are plasmid type of vector (example- c2RB, pJB8)
- Cosmid vectors are hybrids of plasmid and bacteriophage lambda DNA (a small plasmid containing the plasmid origin of replication (ori), an antibiotic resistance gene such as ampR and a suitable restriction site for cloning along with the COS sequences from phage DNA.
- Because of the COS sequence, cosmid recombinants can be packaged into viral particles allowing high efficiency transformation but the distance between two cos sites should be 45-50 kb i.e., if the insert size is smaller than this then it can’t go for packaging.
- Since the recombinant DNA does not encode any lambda proteins, cosmids do not form viral particles (or plaques) but rather forms large circular plasmids and the colonies that arise can be selected on antibiotic plates, like other plasmid DNA recombinants.
- Advantage of cosmids are that they can insert large DNA compared to plasmid and the transformation efficiency is very high as phage can infect E coli very easily.
- The main disadvantage of cosmids is their inability to accept more than 40 – 50 kbp of DNA.
- Some cosmid clones can be unstable during the propagation in E. coli because of the high copy number.
Cosmid Library construction:
- In order to clone a gene of interest into the cosmid, both the gene and cosmid should be cut either with the same restriction enzyme or with two enzymes that will produce identical sticky ends (e.g., BamHI and MboI, as in Fig).
- Note that, the vector is digested two times in order to create cos sides at both ends along with the open up.
- Then mixed in the presence of DNA ligase for the formation of recombinant vector.
- After suitable time interval, the recombinant vectors with DNA fragments, packaged into phages through in vitro packaging method using cos sites of cosmids.
- Then recombinant vectors are transformed to E.Coli with transduction method, the cos end joins forming a circular DNA.
- Transformed E.Coli culture then allowed to grow in a suitable selection medium with ampicillin since vector contains ampicillin resistance gene. For this reason, only the recombinant vector containing colonies grew in the medium.
Advantages of using cosmids as vectors:
- They have high transformation efficiency and they are capable of producing a large number of copies from a small quantity of DNA.
- Also, they can carry up to 45 kb of insertion compared to 25 kb carried by plasmids and λ.
Disadvantages of using cosmids as vectors:
- Cosmids cannot carry more than 50 kb of the insert.