chapter 2: vehicles for gene cloning: plasmids and bacteriophages

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Chapter 2: Vehicles for Gene Cloning: Plasmids and Bacteriophages. DNA DNA 10 Kb DNA . 2.1 . - PowerPoint PPT Presentation

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  • DNADNA10 KbDNAChapter 2: Vehicles for Gene Cloning: Plasmids and Bacteriophages

  • 2.1 2.1.1 (basic features) DNADNADNA

  • A circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA. Plasmids are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells.

  • episome

  • 2.1.2 (size and copy number) 1.0250 Kb DNA

  • 2.1.3 (conjugation and compatibility) conjugative plasmid)(non-conjugative plasmid)

  • 7compatibleincompatibility group :

  • 2.1.4 5 1Ffertility plasmidF

  • oriToriVtra genes32 kb100 kbused to initiate replication for transferused to initiate plasmid replicationIS elements (insertion sequences used in transposition)Discrete region that has transfer genes:tra & trb loci (~40 genes)(Origin of transfer)F plasmid

  • 2RRTFConjugative plasmidTransfer genesR determinantResistance genesTransposons

  • R plasmids code for enzymes that inactivate the drug, prevent the uptake of the drug, or pump out the antibiotic. The resistance factors can be spread throughout an entire colony by way of plasmid transfer, called conjugation. The plasmid contains a tra region which codes for proteins that allow for the passage of the plasmid between cell.

  • 3ColCol plasmidcolicinsColE1 4degradative plasmid)TOL 5virulence plasmidTi

  • 2.1.5 2 m2 m2 m

  • 2.2 2.2.2 bacteriophage, phageDNARNADNARNAcapsid

  • 1DNA 2DNA 3DNA(26

  • 20 minlytic cycleDNADNA

  • 2.2.2 DNADNADNADNAprophage,lysogen

  • M13M13 DNAM13

  • M13 DNA DNA

  • DNA49 KbDNA

  • DNA

  • DNADNA DNADNADNA12DNADNA

  • DNAcohesive endsDNADNAcosDNAcosDNADNA

  • Bacteriophage : cos sequences circularizea linear genome

  • cosrolling circleDNADNADNA(catenane )coscosDNAAcosDNA

  • Rolling circle replicationRolling circle replication generates multiples (concatamers) of the phage genome, which are then processed by the phage nuclease into single genomes.This is an essential intermediate step in viral replication and allows rapid and efficient viral replication.

  • DNA3DNA

  • Consider a circular molecule of double-stranded DNA with a nick in one of the two phosphodiester backbones. As long as there is a free 3' OH end, this can serve as a template for DNA polymerase. When the 3' OH end is extended, the 5' end can be displaced in a manner analogous to the strand displacement reaction. Synthesis on this strand is also analogous to leading strand synthesis. The displaced strand can, in turn, serve as an template for replication as long as a suitable primer is available. Synthesis on this strand is analogous to lagging strand synthesis.If synthesis continues in this manner, the consequence of this mechanism of replication can be the production of concatemer copies of the circular molecule. As a result, multiple copies of a genome are produced.A rolling circle mode of replication is seen both during replication of bacteriophage lambda where rapid production of many copies of the genome is desired, and in the replication of bacteriophage M13 where only a single copy is produced each time.

  • Lambda head genes transcribed and translatedto produce head proteins

  • Endoglucanase A cuts at cossequenceEndoglucanase A expressed. Cuts DNA at cos sequenceand assists packaging lambda DNA into viral capsid(head proteins)

  • Tail genes then expressed

  • Tail bind to heads to form virus

  • Lambda virus produces lysozyme that hydrolysesbacterial cell wall releasing viruses to attack other bacterial cells

  • M13 M13M13 DNADNA,6 407DNAM13315M13

  • M13-----filamentous phage

  • M13 DNADNADNAreplication form, RFDNA100

  • RF DNADNADNAM13DNAM13DNAM131 000

  • Genome:Single stranded circular DNA molecule, covalently closed,6 407 nucleotides10 non overlapping genesHoused in a flexible protein cylinder

  • M13 Gene Functions

  • M13

  • M13 M1310 KbM13M13 M13DNADNAM13

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