Vector construction

It contain basic of vector construction or commally use vector and their some application....

1. Prepared By :- Umang V anani CONSTRUCTION OF V ECTOR

2. WHY IS A VECTOR REQUIRED ? During cloning we transfer a particular gene which has a desired characteristic which we want. For that, we require a vector(vehicle) which helps us to transfer the gene to the bacteria.

3. WHAT IS A VECTOR ? A vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

4. TYPES OF VECTOR 1. CLONING VECTOR 2. EXPRESSION VECTOR

5. EXPRESSION VECTOR The vector design for expression of production protein specified by the DNA insert is called EXPRESSION VECTOR.

6. EXPRESSION VECTOR TWO TYPE :- 1. PROKARYOTIC EXPRESSION VECTOR Bacterial expression system (e.g. E.coli ) 2. EUKARYOTIC EXPRESSION VECTOR Yeast expression system (e.g. S.cervesiae ) Viral expression system (e.g. Baculovirus ) Mammalian cell expression system

7. CLONING VECTORS A cloning vector is a DNA molecule in which foreign DNA can be inserted or integrated and which is further capable of replicating within host cell to produce multiple clone of recombinant DNA.

8. CLONING VECTORS TYPES :- PLASMID BACTERIOPHAGE COSMID BACTERIAL ARTIFICIAL CHROMOSOME YEAST ARTIFICIAL CHROMOSOME HUMAN ARTIFICIAL CHROMOSOME

9. PROKARYOTIC EXPRESSION VECTOR ADVANTAGES :- Gene expression can be easily contolled. Easy to grow with high yields. Easy to manipulate. product can be designed for secretion into the growth media.

10. PLASMID Plasmid vectors can be used for cloning of DNA fragments of size generally ranging from 0.1 to 10 kb. The most common examples of plasmid cloning vectors are pUC18/19, pBluescript, pGEX, pET series etc.

11. PLASMID VECTOR pBR 322 CONTAIN Ampicillin resistance gene. Tetracycline resistance gene. origin of replication . Eco RI site.

12. BACTERIOPHAGE Bacteriophage is a genetically complex but very extensively studied virus of E.coli. The DNA of phage, is a linear duplex molecule of 48502 bp (~49kb) in length.

13. BACTERIOPHAGE The DNA isolated from virus particles is a double stranded linear molecule with short complementary single stranded projections of 12 nucleotides its 5’ ends. These cohesive termini, also referred to as COS site, allow the be DNA to be circularized after infection of the host cell.

14. COSMID Cosmid vectors (5-7 kb) are hybrid between plasmids and phages. They contain antibiotic genes and replication origin from plasmids and ‘cos’ sites from phages which are necessary for packaging of DNA. These vectors can clone larger fragments of size ranging from 35 to 50 kb.

15. LAMBDA PHAGE Lambda phage can also be used as vector for cloning gene of interest of size ranging from 8-25 kb fragments.

16. LAMBDA PHAGE This is based on lambda phage genome which is 48,502 bp with a central 33% (stuffer region) which is not critical for lytic growth and can be replaced with 8-25 kb gene inserts e.g. gt11, charon phages, lambda ZAP vectors. Lambda vectors can be introduced into cells at a very high efficiency.

17. BACTERIAL ARTIFICIAL CHROMOSOME BAC vectors contain sequences from the E. coli F plasmid and they have the ability to clone up to 75 - 200 kb fragments.

18. YEAST ARTIFICIAL CHROMOSOME YAC vectors contain sequences required to replicate and maintain chromosome in budding yeast and contain a yeast origin of replication, a centromere, and a telomere at each end. These vectors are able to clone very large fragments of >2,000 kb inserts (2 Mb).

19. CONSTRUCTION OF VECTOR The first requirement for construction of vector is an extrachromosomal self replicating circular plasmid. A restriction enzyme is required to cut the restriction site where the desired gene is attached. A DNA Ligase is required to join the gene in plasmid at restriction site.

20. VECTOR Four common properties of vector Ability to promote autonomous replication. Contain a genetic marker(usually dominant) for selection. Unique restriction sites to facilitate cloning to insert DNA. Minimum amount of non-essential DNA to optimize cloning.

21. ORIGIN OF REPLICATION Origin of replication is a DNA segment recognized by the cellular DNA replication enzyme. Without replication origin, DNA can not be replicated in the cell.

22. SELECTIVE MARKER Selective marker is required for maintenance of plasmid in the cell. Because of the presence of the selective marker, the plasmid becomes useful for the cell.

23. SELECTIVE MARKER Under selective conditions, only cells that contain plasmid with selectable marker can survive. Gene that confers resistance to various antibiotic are used. Genes that make cells resistance to ampicillin, neomycin or chloramphenicol are used.

24. RESTRICTION ENZYME Mostly Endonuclease is used for cutting of particular selected gene from the DNA and cut the same sequence in the plasmid for the attachment. Two types of cut is done by the enzyme “blunt cut” and “sticky cut”. sticky cut is very specific and unique.

25. INJECTION INTO CELL Applying small heat shock or small electric shock to the cell creates small pores in the cell membrane. Through these small pores, vector is injected in the cell.

26. CHOICE OF EXPRESSION VECTORS Strong promoter Intact ORF Ribosomal binding site Termination sequence

27. CHOICE OF CLONING VECTORS A vector should be small and easily prepared. Capable of autonomous replication in the host cell. It should have one target site only for any restriction enzyme(that has been used in to make DNA fragments. The vector should show Antibiotic resistance. The vector must have a marker permitting recognition.

28. Maximum DNA insert accommodate by different cloning vectors… Types Size of cloned DNA(kb) Plasmid 20 Lambda Phage 25 Cosmid 45 BAC 300 YAC 1000

29. APPLICATION OF VECTOR •The construction of expression libraries. •The analysis of gene function at protein level. •The commercial production of proteins. •The production of antibodies. •For in vivo studies of the protein. EXPRESSION VECTOR

30. APPLICATION OF VECTOR •To amplify the clones of desired DNA segment •Allow in vitro production of RNA copies of desired DNA insert. •Generates single stranded copies of DNA insert •Construction of genomic DNA libraries and cDNA libraries. CLONING VECTOR