Gateway vector.pptx
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The document summarizes Gateway vector technology, which utilizes site-specific recombination to rapidly transfer DNA fragments between vectors. It describes the BP and LR reactions used to clone DNA fragments into entry vectors and then transfer them to destination vectors to generate expression clones. The Gateway system provides an efficient, universal method for multigene delivery and expression in different host systems. Its advantages include speed, compatibility with multiple fragments and host systems, and high cloning efficiency.
Gateway vector.pptx
- 1. Submitted By : Amit Kr Bhardwaj MSc Biotechnology Topic – Gateway Vector Technology
- 2. CONTENTS Introduction Gateway Cloning BP Reaction and LR Reaction Advantages
- 3. Introduction The Gateway Cloning Technology provides a rapid and highly efficient route to protein expression, functional analysis, and cloning/subcloning of DNA segments. Gateway-compatible destination vectors contain the Gateway recombination sites, allowing rapid, efficient transfer of sequences into a variety of expression systems using site-specific recombination. The Gateway cloning system takes advantage of elements that evolved naturally in the life cycle of the bacteriophage lambda (l). During this cycle, the bacteriophage passes from a lysogenic phase, in which the viral genome is stably incorporated into the host genome, to a lytic phase, in which the host cell ruptures (lyses) and infectious phage particles are released.
- 4. The Gateway cloning system utilizes modified att recombination sites, together with an integration enzyme mix containing Integrase (Int) and Integration Host Factor (IHF) proteins (BP clonase) An excision/integratioenzyme mixture containing the Int, IHF, and Excisionase (Xis) proteins (LR clonase), derived from elements used during the bacterio phage l life cycle. DNA fragments flanked by recombination sites can be mixed in vitro with vectors that also contain recombination sites, allowing the exchange of DNA fragments and the generation of recombinant DNA. For Gateway cloning, the att sites have been modified so that the orientation of the DNA fragments can be maintained during the excision and integration process. The PCR fragments flanked by attB1 and attB2 sites to be inserted into pDONR vectors containing the reciprocal attP sites, thereby gen_x0002_erating “entry clones” in which the chosen DNA fragments are flanked by attL1 and attL2 sites. Gateway Cloning
- 5. Figure 1. For lysogeny, the viral DNA is incorporated into the host genome by a process of recombination between common sequences, the att sites, in the two genomes. Bacteriophage has an attP site (P for phage), and the host E. coli DNA contains an attB site (B for bacterium). A number of proteins are required for this recombination: l-derived Integrase (Int) and E. coli–derived Integration Host Factor (IHF) allow l to enter the lysogenic phase of its life cycle, and IHF, Int, and derived Excisionase (Xis) allow l to excise from the E. coli genome and enter the lytic phase of its life cycle.
- 6. BP Reaction & LR Reaction well-characterized DNA fragments for insertion into “destination vectors.” Catalyzed by LR clonase, DNA fragments flanked by attL1 and attL2 sites are then transferred, by a second recombination reaction, to pDEST vectors containing attR1 and attR2 sites. The resulting recombinant DNA constructs are known as “expression clones.” Here, the recombination product of the attL1 and attR1 sites is an attB1 site and the recombination product of the attL2 and attR2 sites is an attB2 site. To select the correct recombination product for the BP and LR reactions, a combination of positive and negative selectable markers are employed. Positive selection is afforded by alternative antibiotic selection, while negative selection is afforded by the ccdB gene, the product of which inhibits the activity of DNA gyrase, leading ultimately to cell death.
- 7. The ultimate goal of the GATEWAY reactions is to make an expression clone. This is often a two step process: Step 1 Cloning the gene of interest into an Entry Vector using the BP Reaction. Step 2 Subcloning the gene of interest from the Entry Clone (Step 1) into a Destination Vector using the LR Reaction producing the Expression Clone.
- 8. BP REACTION CLONING THE GENE OF INTREST INTO AN ENTRY VECTOR incubation for 1 hour attB-PCR product + donar vector + enzyme transform into phage resistant cells colonies are screened by PCR
- 10. LR REACTION SUBCLONING THE GENE OF INTEREST FROM THE ENTRY CLONE INTO A DESTINATION VECTOR TO GENERATE EXPRESSION VECTOR. expression vector + entry clone + enzyme incubation for 1 hour transform into phage resistant cells
- 13. BINARY VECTORS Binary vector is a standard tool in the transformation of higher plants mediated by Agrobacterium tumefaciens. • It is composed of the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, selectable marker genes, reporter genes, and other accessory elements that can improve the efficiency of and/or give further capability to the system. • A super-binary vector carries additional virulence genes from a Ti plasmid, and exhibits very high frequency of transformation, which is valuable for recalcitrant plants such as cereals.
- 15. USES • Optimized multi-gene delivery without transfection. • Expression of enzymatic pathways. • Expression of multisubunit protein complexes. • Gene knock down. • Variable gene expression levels using different expression elements.
- 16. ADVANTAGES • Compatibility and flexibility Once you generate the entry clone with your DNA sequence of interest, you can move this DNA fragment across any expression system in just one recombination step. • Speed The Gateway system enables the generation of the expression construct in only 1 day, as opposed to 2+ days with traditional restriction and ligation cloning. It is also possible to set up the BP and LR reactions in the same tube, speeding up the cloning of the attB-PCR products directly into destination vectors. The cloning process is simple- no restriction, ligation or gel purification steps are required!
- 17. • Multiple fragment cloning You can use Gateway cloning to insert multiple DNA fragments into many vectors at once in the same tube. You can clone up to 4 DNA fragments, in a specific order and orientation, in one tube, into one Gateway vector to produce the desired expression clone. • High efficiency The positive (antibiotic) and negative (CcdB) selection markers used for Gateway Cloning can increase cloning efficiency to >99%. • Universality All types of DNA fragments may be cloned: PCR fragments, cDNA or Genomic DNA and is available for all kind of organisms from mammals to E. coli.
- 22. REFERENCES • Hartley JL. Use of the Gateway System for Protein Expression in Multiple Hosts. Curr Protoc Protein Sci. 2003 Feb;Chapter 5:Unit 5.17. PubMed PMID:18429245. • Ptashne, M. (1992). A Genetic Switch: Phage (Lambda) and Higher Organisms (Cambridge, MA: Cell Press).
- 23. THANK YOU!