Cloning vectors: Definition, Types, Examples, Unit 2 rDNA, PCR Notes
A DNA molecule that is capable of replicating in a host organism and can act as a carrier molecule for the construction of recombinant DNA. Vectors are extra-chromosomal circular DNA molecules which are helpful in transferring the DNA into the host where the desired gene replicates along with host DNA producing desired protein.
A foreign gene cannot be inserted into the host system without a vector
Vectors are basically classified as:
- Naturally occurring vectors
- Artificially synthesized vectors: Designed by biotechnologists in the lab, not naturally occurring.
Types of Cloning vectors
- Plasmid cloning vectors (obtained from bacteria)
- Phage λ cloning vectors (obtained from virus)
- Cosmid cloning vectors (possess a combination of bacteria and virus)
- Shuttle vectors (artificially created)
- Yeast artificial chromosomes (YACS- artificially created from yeast)
- Bacterial artificial chromosomes (BACS- artificially created form bacteria)
Depending on the need and size of gene to be transferred into the particular host a suitable vector is selected. Each vector has its own advantages and disadvantages
Plasmid Cloning Vectors
The bacterial plasmid is naturally occurring, circular double-stranded extra-chromosomal DNA capable of replicating autonomously within a cell.
Autonomous replication means it is dependent on the extra-chromosomal DNA of bacteria for its replication.
Origin sequence (ori) required for replication. Also act as marker for specific plasmid as it is naturally occurring and varies in different micro-organism.
Multiple cloning site (polylinker region) is a site in plasmid where different RE’s can be used to cleave the specific nucleotide sequence which break the phosphodiester bonds. At that site, foreign gene can be inserted. Then the desired gene replicates along with plasmid in the bacteria.
Ampicillin resistant gene (ampR) is a gene added to the plasmid to make the plasmid ampicillin resistant. Since naturally occurring E.coli plasmid is destroyed by ampicillin and hence cannot be taken for r-DNA technology experiments. The Ampicillin gene is used to differentiate between normal and modified plasmids. It is used in r-DNA experiments, only the bacterium which survives when ampicillin is added to the bacterial culture i.e. the one which is modified by adding ampR gene is taken for r-DNA experiments.
Lac Z gene is present near polylinker region produces β-galactosidase enzyme. On insertion of a foreign gene into the polylinker region, the genetic sequence of the plasmid will change and hence β-galactosidase enzyme will not be produced. Since the production of that enzyme requires the Lac Z gene and polylinker region to remain intact (as present in natural plasmid). So it indicates that the foreign gene is inserted at a proper site and the experiment is successful. Hence the proper insertion of the gene in the polylinker region can be determined.
Features of pUC19 (E.g. of Plasmid cloning vector)
- A high copy number in E. coli, ~100 copies/cell, provides a high yield.
- One single E.coli will contain 100 copies of plasmid, so this helps in getting the foreign gene into a particular micro-organism where multiple copies can be produced.
- When the plasmid replicates, the inserted gene will also be multiplied along with it and the product of interest will be obtained. E.g. Insulin gene produces insulin (protein) in E.coli.
- Plasmids may be used to clone the foreign gene of size 15 kb.
- The selectable marker is ampR. Ampicillin in the growth medium prevents the growth of all other normal E. coli.
Phage cloning vectors
- It is not naturally occurring, it’s an Engineered version of bacteriophage (the virus that infects E. coli).
- It is obtained by modifying the characteristics of the virus so it bears some features of the virus and some genes which are artificially added.
- The central region of the chromosome (linear) is cut with a restriction enzyme and digested DNA is inserted.
- DNA is packaged in phage heads to form virus particles.
- Phages with both ends of the chromosome and a 37-52 kb insert replicate by infecting E. coli.
- Phages replicate using E. coli by the lytic cycle
- Produces large quantities of 37-52 kb cloned DNA.
- Like plasmid vectors, a large number of restriction sites are available; useful for larger DNA fragments than pUC19.
- It has the ability to multiply rapidly which is its major feature, so if a large number of copies of foreign genes is required, then a viral vector is used for its replication.
Cosmid cloning vector
- It contains features of both plasmid and phage cloning vectors.
- It has characteristic of plasmid which are useful for carrying foreign gene and also some characteristic of viruses which are useful to increase the capacity (length of gene in terms of base pairs) of this vector.
- Do not occur naturally. It is circular in shape.
- It contains Origin (ori) sequence.
- It contains Selectable marker, e.g. ampR.
- It contains Restriction sites.
- It cos site contains gene obtained from Phage which permits packaging (fragment of DNA) into phages and insertion into E. coli cells.
- Useful for DNA fragment / foreign gene size of 37-52 kb.
- Gene carrying capacity here is more compared to plasmid and phage λ cloning vector.
- This vector can be used in bacteria as well as viruses to transfer genes.
Capable of replicating in two or more types of hosts.
Replicate autonomously, or integrate into the host genome and replicate when the host replicates.
Commonly used for transporting genes from one organism to another (i.e., transforming animal and plant cells).
Example of shuttle vector
- Firefly luciferase gene is inserted into Ti plasmid and transform into Agrobacterium.
- Ti stands for Tumor inducing plasmid obtained from Agrobacteruim tumefaciens which has the ability to infect plants like tobacco. Hence can be successfully used to transfer luciferase gene into tobacco plant.
- Luciferase is an enzyme that produces light by catalyzing the conversion of luciferin to oxyluciferase in the presence of ATP and molecular oxygen.
- The ability of luciferase to produce light has made it an attractive enzyme for use in a wide variety of bioluminescent assays.
- These assays offer high sensitivity of detection, low background and versatility of use
Agrobacterium can be grown in large quantities and when it infects tobacco plant, the plant emits light.
Application of shuttle vectors
Transfering gene from one host to another.
Yeast Artificial Chromosomes (YACs)
Changes are made into genome of naturally occurring yeast chromosome to construct an artificial chromosome.
Eg. Baker’s yeast, Saccharomyces cerevisiae is used
- Yeast telomere at each end ( the artificial chromosome contains genes of yeast at the end of yeast chromosomes)
- Centre of chromosome contains genes of yeast.
- Selectable marker (amino acid dependence, etc.) on each arm.
- Autonomously replicating sequence (ARS) for replication.
- Useful for cloning very large DNA fragments/ foreign gene size up to 500 kb; useful for replication of very large DNA fragments.
- Can carry large number of genes as compared to all vectors discussed above.
Bacterial Artificial Chromosomes (BACs)
Vectors are created as artificial chromosomes which are to be cloned into E. coli.
Obtained from bacteria
Not naturally occurring, changes are made into bacterial chromosome so that it can carry foreign gene.
Useful for cloning DNA fragments/ foreign gene up to 300 kb.
It can be handled like regular bacterial plasmid vectors.
Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects.
Like other vectors, BACs contain:
Origin (ori) sequence derived from an E. coli plasmid called the F factor.
Multiple cloning sites (restriction sites).
Selectable markers (antibiotic resistance).
BAC’s are based on F plasmid, which is much larger than standard plasmid cloning vector and therefore offers the potential of cloning large fragments.
Most of the sequencing of human genome has been accomplished using a library of BAC’s recombinant.
SIMPLIFIED CONCEPTS OF BIOTECHNOLOGY By, Dr. Pramod Kadu & Ms. Suchita Vishwakarma
Third Year B Pharm Notes, Syllabus, Books, PDF Subjectwise/Topicwise