PUc19 plasmid cloning vector genetic engineering ppt
- 2. content • Plasmid and it's types • Plasmid as vector • pUC19 vector • The construction of pUC vectors • Steps of making recombinant • Restriction • Ligation • Transformation • Selection • Advantages of pUC Vectors 2
- 3. Plasmid and it's types Plasmids are self-replicating, double-stranded, circular DNA molecules that are maintained in bacteria as independent extrachromosomal entities. Types of Plasmids Conjugative Plasmids/ Fertility plasmid /F Plasmid - Involved in the process of conjugation R Plasmids (Resistance Plasmids) -Carry genes that confer resistance to antibiotics or toxins. Metabolic Plasmids - Contain genes that enable the degradation of unusual compounds, allowing bacteria to utilize diverse substrates. Cryptic Plasmids - Have unknown or non-functional genes their role in the host is often unclear. range in size from less than 1 kb to more than 500 kb. Each plasmid has a sequence that functions as an origin of DNA replication. 10 to 100 copies per host cell – high copy number plasmids. 1 to 4 copies per cell - low-copy-number plasmids. 3
- 4. Plasmid as vector Vector –They carriers foreign DNA to the host cell work as a vehicle. Cloning vector – Making copies of DNA. (Replication) example - pUC19, pBR322 Expression vector – production of Proteins (translation) example- pET , pGEX Quality of vectors Small in size , high copy no. It must be containing unique restriction sites It must have one or more selectable marker gene capable of autonomously replicating inside the host for identifying recipient cells 4
- 5. pUC19 vector pUC are obtained by modifying the pBR322 vector. pUC vectors are smaller than pBR322 of being only ~2.7 kb 2686. But comparatively they have a high copy number. 500 to 600 copies of the plasmid per cell. The Nomenclature of pUC Vectors: 'p' indicates the plasmid. 'UC' stands for university of California where it was first developed by J. Messing et al. 19 numerical designation 5
- 6. The construction of pUC vectors • Origin of Replication • Selectable Marker • multiple cloning sites (e.g., EcoRI,SacI, KpnI, XmaI, SmaI, BamHI, XbaI, SalI, HincII, AccI, BspMI, PstI, SphI,and HindIII), which is called a multiple cloning site (or polylinker) 6
- 7. Steps of making recombinant puc19 vector and cloning it in host cell Restriction Ligation Selection Transformation 7
- 8. Restriction • Purified, closed circular pUC19 molecules are cut with a restriction enzyme that lies within LAC Z GENE cleaves the plasmid DNA only once to create single, linear, sticky-ended DNA molecules • These linear molecules are combined with prepared target DNA from a source organism. This DNA has been cut with the same restriction enzyme, which generates the same sticky ends as those on the plasmid DNA 8
- 9. Ligation After the restriction digestion DNA mixture treated with T4 DNA ligase in the presence of ATP. Under these conditions, number of different ligated combinations are produced Original closed vector joining of the target DNA Recircularization of vector Joining of the Target DNA to a vector. 9
- 10. Use of enzyme alkaline phosphatase • plasmid DNA preparation is treated with the enzyme alkaline phosphatase to remove the 5′ phosphate groups from the linearized plasmid DNA. • Now T4DNA ligase cannot join the ends of the dephosphorylated linear plasmid DNA. • However, The source DNA, which provides the phosphate groups, are sufficient to hold the two molecules together, despite the presence of two nicks After transformation, these nicks are sealed by the host cell DNA ligase system. 10
- 11. Transformation Uptake of foreign DNA by host cell is called Transformation. Transformation is done by Electroporation. For Ecoli. ~50ul cells 25 microfarads,2.5 kilovolts and 200 ohm for ~ 4.6 milliseconds are suitable. Transformation is a less efficient technique. 11
- 12. Selection • Selection It is the strategy for selecting host cell that have been transformed with pUC19(loaded vector).The transformation contain 3 cell type:- • Non transformed cell • Cells with the intact original plasmid. • Cell with target DNA cloned into the restriction site of pUC19 (recombinant plasmid) pUC19 GOI Loaded vector 12
- 13. host cells are plated onto medium that contains ampicillin Non-transformed cells cannot grow in the presence of ampicillin. When cells carrying unmodified pUC19 are grown in the presence of Inducer isopropyl-β-d-thiogalactopyranoside (IPTG). lacI gene - Repressor can no longer bind to the promoter–operator region of the lacZ′ gene. LacZ gene- form β-galactosidase. In pUC19, the multiple cloning site is incorporated into the lacZ′ gene in the plasmid which inactivates it Substrate- 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) it hydrolyzed by β-galactosidase to a blue product. Under these conditions, colonies containing unmodified pUC19 appear blue 13
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- 15. Selection of host with loaded vector ( Blue white screening) host cells are plated onto medium that contains ampicillin, IPTG, and X-Gal. Non-transformed cells cannot grow in the presence of ampicillin. Cells with non-recombinant puc19 plasmids can grow with ampicillin in the medium, and because they can form functional β-galactosidase, they produce blue colonies. In contrast, host cells that carry a plasmid–cloned DNA construct produce white colonies on the same medium. The reason for this is that, usually, DNA inserted into a restriction endonuclease site within the multiple cloning site disrupts the correct sequence of DNA codons (reading frame) of the lacZ′ gene and prevents the production of a functional LacZ′ protein, so no active β-galactosidase is produced . In the absence of β-galactosidase activity the X-Gal in the medium is not converted to the blue compound, so these colonies remain white. 15
- 16. Advantages of pUC Vectors: • High copy number of 500-600 copies per cell • Easy and single step selection. • Small size Disadvantages of pUC19: • It cannot accommodate a gene of interest larger than 15kb. 16
- 17. Conclusion pUC19 is a valuable cloning vector widely utilized in molecular biology for its compact size, high-copy number replication in bacterial hosts, multiple cloning sites (MCS) within the lacZ gene, and the presence of selectable markers such as ampicillin resistance. Its utility extends to applications like blue-white screening making it a important in genetic engineering and molecular cloning experiments. 17
- 18. REFERENCES • Glick –recombination biotechnology • TA Brown(2005). Gene cloning and DNA analysis.4th edition • Principle of gene manipulation and genomics by Old and primrose (2001) 18
- 19. THANKYOU 19