- Capable of replicating inside the host.
- Have compatible restriction site for insertion of DNA molecule (insert).
- Capable of autonomous replication inside the host (ori site).
- Smaller in size and able to incorporate larger insert size.
- Have a selectable marker for screening of recombinant organism.
Properties of good host:-
- Be easy to transform.
- Support the replication of recombinant DNA.
- Be free from elements that interfere with replication of recombinant DNA.
- Lack active restriction enzymes, e.g., E.coli K12 substrain HB 101.
- Should not have methylases, since, these enzymes would methylate the replicated recombinant DNA
which, as a result, would become resistant to useful restriction enzymes.
- Be deficient in normal recombinant function, so, that, the DNA insert is not altered by recombination
events.
> In 1952, the term plasmid was introduced by Joshua Lederberg, the American molecular biologist to refer to “any extrachromosomal hereditary determinant”.
> A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently.
Characteristics of ideal plasmid vectors:-
i. Size:- Plasmid must be small in size. The small is helpful for easy uptake of cDNA by host cells and for the isolation of plasmid without damage. Ideal vector should be less than or equal to 10kb. The small size is essential for easy introduction in cell by transformation, transduction and electroporation.
ii. Copy number:- The plasmid must be present in multiple copies.
iii. Genetic markers:- Plasmid must have one or few genetic markers. These markers help us for the selection of organism that has recombinant DNA
iv. Origin of replication:- The plasmid must have its own orogin of replication and regulatory genes for the self-replication.
v. Unique restriction sites:- The plasmid must have unique restriction sites common restriction enzymes in use.
vi. Multiple cloning sites:- This property permits the insertion of gene of interest and plasmid recircularization.
vii. Insertional inactivation:- The plasmid must have unique sites for restriction enzymes in marker genes. This will help us for the selection of recombination by insertional inactivation method.
viii. Pathogenicity:- The plasmid should not have any pathogenic property.
ix. Should not be transferred by conjugation:- This property of vector molecule prevents recombinant DNA to escape to natural population of bacteria.
x. Selectable make gene:- Vector molecules should have some detectable traits. These traits enable the
transformed cells to be identified among the non-transformed ones. eg. antibiotic resistance gene.
Examples:-
a. pBR322:-
- pBR322 is a widely-used E. coli cloning vector.
- It was created in 1977 in the laboratory of Herbert Boyer at the University of California San Francisco.
- The p stands for "plasmid" and BR for "Bolivar" and "Rodriguez", researchers who constructed it.
- pBR322 is 4363 base pairs in length.
- pBR322 plasmid has the following elements:-
i. “rep” replicon from plasmid pMB1 which is responsible for replication of the plasmid.
ii. “rop” gene encoding Rop protein, are associated with stability of plasmid and also controls copy number (increase number). The source of “rop” gene is pMB1plasmid.
iii. “tet” gene encoding tetracycline resistance derived from pSC101 plasmid.
iv. “bla” gene encoding β lactamase which provide ampicillin resistance (source: transposon Tn3).
- It carries two sets of antibiotic resistance genes. Either ampicillin or tetracycline resistance can be used as a selectable marker for cells containing the plasmid.
- It has a reasonably high copy number. Generally, there are about 15 molecules present in a transformed E. coli cell, but this number can be increased up to between 1000 and 3000 by plasmid amplification in the presence of a protein synthesis inhibitor such as chloramphenicol.
- It has 528 restriction sites for 66 restriction enzymes. Among them 20 restriction enzymes cut it at
unique restriction sites. Tetracycline has 6 unique sites for 6 restriction enzymes. Ampicillin gene has 3
unique restriction site.
b. pUC plasmids:-
- pUC plasmids are small, high copy number plasmids of size 2686bp.
- This series of cloning vectors were developed by Messing and co-workers in the University of California.
- The p in its name stands for plasmid and UC represents the University of California.
- pUC vectors contain a lacZ sequence and multiple cloning site (MCS) within lacZ. This helps in use of broad spectrum of restriction endonucleases and permits rapid visual detection of an insert.
- pUC18 and pUC19 vectors are identical apart from the fact that the MCS is arranged in opposite orientation.
- pUC vectors consists of following elements:
i. “rep” replicon region derived from plasmid pBR322 with single point mutation (to increase copy
number).
ii. “bla” gene encoding β lactamase which provide ampicillin resistance which is derived from pBR322. This site is different from pBR322 by two point mutations.
iii. E.coli lac operon system.
- “rop” gene is removed from this vector which leads to an increase in copy number.
2. Cosmids:-
> It is first described by Collins and Hohn in 1978.
> It is formed by joining ends of a linearized plasmid DNA with cos-site of lambda DNA.
> It is the commonly used cloning vector suitable for cloning large DNA fragments upto 45 kbp.
> Cosmid has an origin of replication, selectable markers, and gene cloning sites of plasmid DNA.
Salient features:-
i. Cosmid is a circular ds DNA.
ii. It has two complementary single-stranded regions at both ends of a plasmid DNA. The two cos-ends form a duplex by base pairing.
iii. The cosmid DNA does not code for phage proteins and host cell lysis.
iv. It does not involve in multiplication of phage particles.
v. It has an origin of replication from plasmid DNA for independent replication.
vi. It has selectable marker genes and gene cloning sites of plasmid DNA
vii. The cosmid DNA is packed within protein coat of bacteriophage to form inactive phage particles. Cos-site is a prerequsites for invitro packaging of cosmid in phage protein coat.
viii. After infection, the cosmid DNA does not integrate into host chromosomal DNA. It exits as a definite extra chromosomal DNA and replicates independently.
Examples:-
i. Cosmid pLFR5:-
- It is 6 kbp in size.
- It is constructed from E.coli plasmid pBR322 and two cos-ends of lambda DNA.
ii. Cosmid pJB8:-
- It is 5.4 kbp in size.
- It is constructed from the plasmid pBR322 and cos sites of lambda DNA.
iii. Cosmid pHC79:-
- It is 6.5 kbp in size.
- It is constructed from pBR322 and cos-sites of lambda DNA.
Advantages:-
- Cosmid pick up relatively larger DNA fragments than the plasmid do.
- As cosmids pick up large DNA fragments, they are used to establish gene libraries
- Gene cloning through cosmids helps in the study of non-sence sequences in the genome of organisms.
- Some cosmids are constructed by joining a linearized plasmid DNA with DNA fragments of p1
bacteriophage that have cos-ends. The P1 bacteriophage has the genome of 115 kbp. So, a DNA of 85
kbp can be packaged into the head of P1 phage. These cosmids help to clone large genes and gene
clusters in bacteria.
Disadvantages:-
- The packaging enzyme fails to pack recombinant cosmids into the phage head, if any one of the two
cos-ends is missing.
- Sometimes more than one recombinant cosmid join together to form a large DNA. If so, the packaging
enzyme fails to pack the DNA into the phage head.
- Slower replication
- Higher frequency of recombination inside bacterial host.
- Unstable inside E.coli host and thus easy to lose vector.
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