Vectors in Recombenant DNA technology

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1. Vectors M.Kamil khan Microbiologist

2. Introduction In  molecular cloning , a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material (target DNA having gene of interest into another  cell , (host cell i.e E.coli ) where it can be replicated and expressed. A vector containing foreign DNA is termed  recombinant DNA .

3. Property of vectors The genetic martial or DNA should be called as vectors when they have the following properties. Multiple Cloning sites. Origin of replication and promoter. Start and stop codon. Easily isolate and transfer to the host cells. Selectable marker genes. Small molecular size and weight.

4. Types of vectors Expression vectors. Cloning vectors. Plasmid. Cosmid . Phage vector. Bacterial artificial chromosomes (BAC). Yeast artificial chromosomes (YAC).

5. Cloning vectors A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

6. 1. Plasmid Plasmids   are double-stranded extra chromosomal and generally circular DNA sequences that are capable of replication using the host cell's replication machinery.  Plasmid vectors minimalistically consist of an  origin of replication  that allows for semi-independent replication of the plasmid in the host. Plasmids vectors are natural and found in many bacteria, for example in  E. coli , a few eukaryotes, for example in yeast such as  Saccharomyces cerevisiae .

7. Size range 25 kb. Examples of plasmid pBR322 pBR327 pBR325 pBR328 pUC8 pUC9 pUC12 pUC13 pGEM3Z

8. THREE TYPES OF PLASMID 1. Fertility plasmids:- can perform conjugation. 2. Resistance plasmids:- contain genes that build a resistance against antibiotics or poisons. 3. Col plasmids:- contain genes that code for proteins that can kill bacteria.

9. Advantages of plasmid Present in a high number inside cells Having already selectable markers Fastly replicate Having small size and can be handle Easily isolate and manipulate Easy purified Disadvantages of plasmid Only 12 kb size foreign DNA can be cloned Require competency Expensive Self anneal

10. 2. Bacteriophage lemda Naturally Approximately 50 kb in length Modification should perform to make it suitable for utilization ( lytic 35kb lysogenic 15) Two types Insertion vectors Replacement vectors

11. Advantages Having cos site due to which it can package the DNA in phage head Can prepare cDNA and gene libraries Use to clone large fragment of DNA(10-20kb). Disadvantages Only carry 20 kb foreign DNA Less use to handle

12. 3. Cosmid Modified and artificial version of vectors. Hybrid vectors because it contain lemda phage sequences (cos site +plasmid= cosmid ). Can carry 45 kb target DNA. Having cos site for packaging the DNA .

13. Advantages Build up genomic libraries Can carry 45 kb of DNA Disadvantages Unstable Difficult to maintain Can only clone 45kb foreign DNA

14. 4 . Bacterial artificial chromosomes Can carry 50-100 kb foreign DNA used for the analysis of genomes Examples pBACe3.6, pBAC11.

15. Advantages Easily manipulate can diagnose genetic disease due to transgenic mice experimentation Can also diagnose neurological disease like Alzheimer disease Disadvantages Can only carry 50-100kb foreign DNA Mutation

16. 5 . Yeast artificial chromosomes (YACS) Linear DNA vectors that resembles normal yeast chromosomes Contain telomere and centromere Can carry 1mb foreign DNA Artificially synthesized

17. Advantages Can clone large fragment of DNA i.e 500kb to 1mb Disadvantages Very fragile and prone to breakage Difficult to separate from the other host cell Unstable and sometime the foreign DNA deleted

18. Expression vectors A plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.

19. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein.  The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively.   Escherichia coli  is commonly used as the host for protein production, but other cell types may also be used. An example of the use of expression vector is the production of insulin, which is used for medical treatments of diabetes.

20. Elements in expression vectors The expression vector should have all cloning vector properties and they have also the following elements for protein expression. Strong promoter Translation initiation sequences ( having ribosomal subunit site and start codon) Translation ter mination sequences (having stop codon) Tag sequences i.e histadine

21. Thanks