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Hosts & Vector

Hosts & Vector. Selecting Cells with Plasmid Vector. Many cells will not take up plasmid during transformation Cells with plasmid can be identified because original plasmid contained gene for antibiotic resistance (ampicillin)

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Hosts & Vector

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  1. Hosts & Vector

  2. Selecting Cells with Plasmid Vector • Many cells will not take up plasmid during transformation • Cells with plasmid can be identified because original plasmid contained gene for antibiotic resistance (ampicillin) • Use medium with ampicillin – if bacteria grow then plasmid must be present • But don’t know if plasmid had DNA insert

  3. Selecting Cells with Plasmid that Carries DNA Insert • Use beta-galactosidase system to do blue-white screening • Host must lack enzyme and plasmid vector must carry gene for beta-gal • Restriction site is within beta-gal gene • Thus, if insert occurred within beta-gal gene, enzyme not produced • X-gal = artificial substrate added to medium that turns blue if enzyme present, otherwise bacteria are normal white color

  4. Cloning Considerations • Choosing a vector • Plasmids limited to small molecules • Bacteriophages (phages) are viruses that infect bacteria • Phages can carry DNA inserts up to 15,000 nucleotides long

  5. Cloning Considerations Choosing a host • Bacteria very good, but limitations • Bacteria lack ability to modify proteins and limited size of insert • Yeast (Saccharomyces cere isiae) excellent for many applications • Occasionally necessary to clone genes into specific animal or plant hosts – more difficult but possible

  6. Hosts & Vectors • Host systems: - Bacterium (E.coli) - Yeast (Saccharomyces cerevisea) - Insect cells - Mammalian (Chinese Hamster Ovary cells) • Cloning vectors - derived from natural replicons - Capable of replicating and isolation from host. - Contain a selectable marker to distinguish host cells containing the vector from amongst those that do not (eg. antibiotic resistancy or survival under certain growth conditions.

  7. Types of Vectors • Plasmid DNA E. coli vectors, extra-chromosomal and circular • Bacteriophages Phage l – clone large DNA fragments and incorporate into host genome Phage M13 – allows cloned DNA to be isolated in single-stranded form • Cosmids hybrids of plasmid-bacteriophage l • Artificial chromosomes - Cloning of very large genomic fragments - BACs (bacterial artificial chromosomes) - YACs (yeast artificial chromosomes

  8. Types of Vectors

  9. Vectors used in different Hosts • Bacteria E. coli cloning and expression vectors eg. pGEMT from Promega; pGEX from Invitrogen pQE from Qiagen • Yeast yeast episomal plasmids for gene expression eg. PICHIA expression vectors from Invitrogen • Plants Agrobacterium tumefaciens Ti plasmid introduce genes into plants • Eukaryotic cells Plasmid vectors used for gene expression and functional studies eg. Viruses – SV 40, baculovirus, retroviruses

  10. BacteriophageCloning Vectors

  11. http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gifhttp://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif l Vector DNA - Bacteriophage • viruses that infect bacteria • known dsDNA sequence of ~ 50 kb • linear double-stranded molecule with single-stranded complementary ends • cohesive termini (cos region)

  12. l Vector DNA - Bacteriophage Desirable properties of λ phage: • can accept large pieces of foreign DNA • tremendous improvement over the years • can be reconstituted in vitro

  13. Bacteriophage l • l phage genome - linear 48.5 kb genome. • Each ends consists of cos (cohesive) sites – 12 bp cos ends Cos ends allows DNA circularization in the cell • Central region of genome are non-essential portions and can be replaced byforeign DNA (up to 23kb)

  14. Bacteriophagel

  15. Phage particles injects linear DNA into the cell • DNA ligate to form circle • Replicate to form many new phage particles which are released by cell lysis and cell death • or DNA intergrate to host genome by site-specific recombination (lysogenic phase)

  16. Lysis plaques of l phage onE. coli bacteria. bacteria lawn plaques l bacteriophage Plaques: the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.

  17. M13 phage vectors • Replication form (RF, dsDNA) of M13 phage can be purified and manipulated like a plasmid. • Phage particles (ssDNA): DNA can be isolated in a single-stranded form • DNA sequencing • Site-directed mutagenesis Cloning (RF, like plasmid)  transfection (recombinant DNA)  growth (plating on a cell lawn)  plaques formation (slow growth)

  18. Hybrid plasmid-M13 vectors • Small plasmid vectors (pBluescript) being developed to incorporate M13 functionality • Contain both the plasmid and M13 origin of replication • Normally propagate as true plasmids • Can be induced to form single-stranded phage particles by infection of the host cell with a helper phage.

  19. M13 phage • M13 phage contains a circular 6.7kb ssDNA • Replicate in E. coli cells as double-stranded circles (replicative form, RF), ~ 100 copies per cell • Cells are not lysed by M13, but grow slowly. • Recombinant M13 phage can produce either - dsDNA RF can be isolated & manipulated as plasmid - ssDNA isolated from phage particles in growth medium ( used for DNA sequencing and site-directed mutagenesis)

  20. M13 phage cloning vectors • M13 RF containing cloned fragment (eg. M13amp18 and 19) - Transfect into E.coli cells - plating in a lawn of cells produce plaques - Plaques consist of slow growth rather than lysis of infected cells - Blue-white selection using MCS and lacZ • Hybrid plasmid – M13 vectors (eg. pBlueScript) - eveloped to incorporate M13 functionality - contain plasmid & M13 origin of replication, minus the genes for full phage life cycle. - propagate as true plasmid - can be induced to form single-stranded phage particles by infection of the host cell with a helper phage, provides the gene products required for ss production and packaging

  21. Cosmid vectors • Utilizing the properties of the phage l cos sites in a plasmid vector. • A combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the l head. • The insert can be 37-52 kb.

  22. Formation of a cosmid clone Digestion Ligation

  23. Advantages : Vector DNA - Plasmids • small circular dsDNA that autonomously replicates apart from the chromosome of the host cell • “molecular parasites” • carry one or more genes some of which confer resistance to certain antibiotics • origin of replication (ORI) --- a region of DNA that allows multiplication of the plasmid within the host • plasmid replication: stringent or relaxed

  24. Advantages : Vector DNA - Plasmids Desirable properties of plasmids: • small size • known DNA sequence • high copy number • a selectable marker • a second selectable gene • large number of unique restriction sites

  25. Vector DNA - Plasmids http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif

  26. Vector DNA - Plasmids

  27. Vector DNA - Cosmids • modified plasmids containing cos sequences • carry an ORI & an antibiotic resistance marker • can accommodate ~35 to 45 kb of foreign DNA • can be propagated as plasmids • can be introduced into host by standard procedures • chief technical problems occur when used for library construction

  28. Still remember transformation? JUST A SUMMARY !!!

  29. http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/ligation.gifhttp://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/ligation.gif Inserting the DNA into the vector • Means of inserting foreign DNA into the vector Ligation of the DNA into the linearized vector Requirements for a ligation reaction: • two or more fragments of DNA (blunt/cohesive) • buffer containing ATP • T4 DNA ligase

  30. Transfer of DNA into the host cell • Method of placing the in vitro modified DNA into the host cell Transformation into the host cell • bacterial cells take up naked DNA molecules • cells are made “competent” • cells treated with ice-cold CaCl2 then heat-shocked • efficiency of 107 to 108 transformed colonies/μg DNA • maximum transformation frequency of 10-3

  31. http://bme.pe.u-tokyo.ac.jp/research/ep/img/electroporation.jpghttp://bme.pe.u-tokyo.ac.jp/research/ep/img/electroporation.jpg Transfer of DNA into the host cell Electroporation of the DNA into the host cell • “electric field-mediated membrane permeabilization” • high strength electric field in the presence of DNA • protocols differ for various species • efficiencies of 109 per μg DNA (3 kb) & 106 (136 kb)

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