Vector isolation
- 1. Vectors and vector Isolation By Aqsa
- 2. Content • Vectors • Types of vector 1. Cloning vector 2. Expression vector • Vector isolation
- 3. Origin • From Latin word “Vehere” • Mean carrier . Have different definition in different fields of science. In Biology, It is an organism that acts a vehicle to carry pathogen to the host
- 4. Vector In biotechnology It is a vehicle that act as a carrier for transmission of gene of interest or foreign DNA into organism and possess self-replicating properties. pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer
- 5. Why vector is required? • To transfer a gene having desired and particular characteristics. • For the transmission of the healthy gene. • Vectors to be chosen is dependent on the size and the objective, for which purpose we are cloning.
- 7. It is Properties of vector of low molecular weight and have size less than 11KB able to self-replicate. Possess suitable marker genes to help in recognition of cell that contain vector specific target and recognition sites for various enzymes Have capability of incorporating either itself or DNA into host genome. origin of replication to ensure replication. Able to isolate and purify. Introduce into host cells
- 8. Classification of vectors (On the basis of) Gene of interest Cloning vector Expression vector Host Cell for Bacteria Viruses Animals Plants Cellular Nature Prokaryotic Eukaryotic
- 9. Imp components of a vector Origin of replication Multiple cloning site Antibiotic resistance (selectable marker)
- 10. Types of vectors • It is DNA fragment capable of replication and is stable carrying another DNA fragment (foreign) to insert or introduce it into cell. Cloning vector • It possess all properties of cloning vectors and also have sites where transcription ad translation can occur. Expression vector
- 11. Cloning vs expression vectors Cloning vectors • Only replication Contain Origin of replication, Poly linker & Antibiotic resistance e.g. Plasmid, cosmids, phagemids Expression vector • Replication and transcription • Contain promoter, tag gene and stop and start sequences • Adenoviral vectors
- 12. Cloning vectors • Plasmids, cosmids, phagemid, λ (insertion and replacement), BAC and many others are used as cloning vectors • Use • Cloning vectors are used for Genomic library formation • In genetic engineering for making probes use in the experiments and studies.
- 13. Plasmid • Circular and naturally present DNA in bacteria. • Fragment of extra chromosomal DNA that is stable after being inherited from one to next generation and do not depend on cell cycle for replication. • They possess restriction site to allow DNA (foreign) to incorporate and also have origin for replication. • Conjugative and Non conjugative.
- 14. Bacteriophage • Viruses that infect bacteria • Intracellular obligate parasites that multiply inside bacterial cell by making use of some or all of the host enzymes. • Bacteriophages have a very high significant mechanism for delivering its genome into bacterial cell • Most of genome is can be replaced with foreign DNA. • A DNA fragment of size up to 20 kb can be transformed.
- 16. Cosmids • B. Hohn and J. Collins are known for the development of cosmids in 1978. • Empty protein coat of virus package with the plasmid are cosmids and is transferred with the help of infection and carry double amount of DNA as compared to phage.
- 17. Viral vectors • Eukaryotic cells can be the subject of viral vectors used to transfer gene. • Some organism cannot be processed without viral vectors and they also increase expression of gene that has been transduced. • Adenovirus is used mostly as viral vectors. • They are use in both gene therapy and vaccine development.
- 18. Phagemids • They are better vectors than phages containing F1 phage to produce DNA consisting of single strand . • It is the combination of Plasmid and phage sequences.
- 19. Bacterial artificial chromosome • Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to clone very large DNA fragments ranging in size from 75 to 300 kb.
- 20. Expression vector • Uses They are used when there is a need for the purification of products of genes in large amount Preparation of RNA probe.
- 21. Yest artificial chromosome • YACs are yeast expression vectors. • A very large DNA fragments whose sizes ranging from 100 kb to 3000 kb can be cloned using YACs. • Mostly YACs are used for cloning very large DNA fragments and for the physical mapping of complex genomes.
- 22. Other vectors Plant cell vector • Vectors are also present to transfer the genes in plants cell such as Ti Vector acquire from Agrobacterium tumefaciens. • The purpose of this transfer is to make the plants resistance against certain chemical e.g., insecticide, pesticide etc.
- 23. Vector isolation • Different techniques such as • Gel electrophoresis, • Centrifugation • Column chromatography are involved.
- 25. Isolation of plasmids • Alkaline lysis • Plasmid from the targeted bacteria is obtain by its lysis. • Done by two chemicals Sodium Dodecyl Sulphate and sodium hydroxide. • For the disruption of maintaining structure of cell. • Membrane and proteins are destroyed
- 26. Steps involved 1.Bacterial cultivation 2.Lysate formation by SDS & NaOH 3.Resuspension by buffer 4.Neutralization by Potassium acetate 5.Precipitation / centrifugation 6.Washing by ethanolic buffer 7.Elution 8.Gel Electrophoresis
- 29. Procedure Media suited for the growth of bacteria from which plasmid is obtained is Luria Bertani medium. It increases the plasmid yield. Bacteria are cultured in LB medium. Then their lysis is done by alkaline medium such as (SDS) and NaOH to disrupt structure and membrane of cell. After lysis of cell the product is plasmid with chromosomal RNA (remove after with RNase) and DNA (removed after with the help of centrifugation by pellet formation which is the removed).
- 30. As a result of lysis two function has been performed first is in water the breaking of H bond and breaking of bond between nucleic acids and water. Resuspension buffer help in the isolation pf plasmid from bacteria such as E. coli. Neutralization is then done by the chemical (potassium acetate) which led to the rejoining of the strands of DNA (plasmid). Centrifugation cause the formation of supernatant and separation of other cell components and debris by precipitation or pellet formation. Loading of supernatant onto the column is done. The step of washing then take place to remove impurities with ethanolic washing. After this, pure plasmid is isolated. Next step is the analysis of Plasmid by Gel electrophoresis in which different DNA will be separated due to difference in their charge.
- 31. Plasmid by Gel electrophoresis
- 32. Thank You