Lambda vector
- 1. Phage Based Vector by Dr K. K.Gupta
- 2. • Plasmid has carrying capacity of maximum 10 kb insert • For DNA library DNA size needed is large • So virus is used as vector • viral vectors are genetically engineered to carry the cloned genomic DNA /c DNA • The basis of gene introduction into the host cells lies in the process of Transduction via which a virus infect and inject the viral DNA
- 3. Natural Lambda Phage • λPhage is a temperate phage that infects E.coli and replicates by lytic & lysogenic cycle . 100 phage is released after lysis
- 4. Circularization of Lambda Genome in to the host Bacteria
- 6. FEATURES At either end bears 12 nucleotides stretch with cohesive end /sticky end called Cos sites. It plays important role in two ways :- Helps in recircularizations inside host cell In lytic cycle it forms catenate i.e a linear series of replicated DNA join together by Cos site. This site act as recognition Genome -single linear dS DNA of about 50 kb(The genome contains 48,490 base pairs) and about 20 % is essential for exision and integration (I/E ) events so 20 Kb can be replaced by insert to be used as vector Has 50 gene and 5o% are needed for infection
- 7. λ-Bacteriophage replicates by a rolling circle mechanism before lytic cycle and the cos sites helps in recognition by RE, resulting in to concatameric molecules composed of several linearly arranged recombinants. Just before packaging of the lambda DNA in to capsid, it cleaved at cos site and a single Lambda DNA packaged into capsid
- 8. Cos sites
- 9. Molecular aspects of Lytic cycle • An infective lambda phage has tubular protein tail and protein head enclosing 50 kb DNA . • If less than 50 kb like 38 kb , the viral DNA is packed, it becomes non infective or cannot be packed • In contrast more than 52 Kb can not be packed. Presence of cos allows correct packaging due to presence of endonuclease enzymes at opening of head. • By assembling empty phage particle and 50 kb DNA and tail assemblies ,infective particles can be produced.
- 10. Continued………….. • There are two Bam site that flank the I/E region • When digested with Bam HI three sements are formed • Left segment –contain genetic information for Head & Tail • Right segment – for replication & Lysis • Middle- for I/E ( Integration & Excision ) • Middle piece is replaced by DNA insert of 20 Kb
- 13. Process for creation of λ Vector • Cutting of Source DNA with Bam HI • Isolation of !5-20 Kb DNA fragments • Ligation with the T4 Ligase of L +R and this segment • Added Empty bacteriophage , Tail • 50 Kb DNA is packed to form infective phage to be acted as vector >52 Kb <398 Kb can not be packed . Recombinant Bacteriophage is introduced into host E, coli
- 14. In-vivo packaging • In the in vivo packaging of λ DNA, first the pre-heads are made, these preheads are the major capsid protein encoded by gene E. • After synthesis of preheads the single λ DNA molecules are inserted into each pre-heads. • These single λ DNA molecules are prepared by cutting of concatamerized λ genomes at each cos sites. • The maturation of preheads are done by insertion of a minor protein named D to complete the head and the products of other genes serve as assembly proteins, ensuring joining of the completed tails to the completed heads.
- 15. In vitro packaging • In-vitro packaging of λ with the use of helper phage • The in-vitro packaging of λ takes place by utilizing two E. coli strains having λ lysogens that have several defects in the genes of pathway responsible for packaging. • Due to mutation in gene responsible for production of protein E, prevents preheads being produced in strain BHB2688 (helper phage). • In strain BHB2690 (helper phage), mutation in gene D prevent maturation of the preheads, with packaged DNA, into complete heads. • The functional parts of the BHB2688/BHB2690 mixed lysate having all the components and provide all the products for correct packaging, complementing each other’s deficiencies. • Accordingly, recombinant λ genomes is being constructed in vitro and enclosed into mature λ phage particles before being propagated and replicated in host E. coli cells
- 17. Screening of Bacteriophage DNA libraries • by DNA probes • Immunological assays • The lytic cycle produced plaques due viral zone of lysis and contain bacteriophage • It is lifted on matrix and processed • For DNA hybridization .the proteins are removed , DNA denaured and bound to matrix . Probe of known gen is added to identify the insert. • Immunological assays proteins encoded by cloned gene during lytic cycle is synthesised.These protein are transferred along with plaque and subesquently bound to matrix • Positive response plaques are selected
- 18. By DNA probe Hybridization
- 19. Screening by Immuno assay • The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. • The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. • The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. • The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. • Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization.
- 20. Immunoassay
- 21. Thanks