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Publicada porFidelia Ponce Modificado hace 9 años
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Víctor de Lorenzo y Marc Valls Centro Nacional Biotecnología, Madrid
Herramientas genéticas en microbiología ambiental III Mini-transposones Víctor de Lorenzo y Marc Valls Centro Nacional Biotecnología, Madrid
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Insertion sequences vs. transposons
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Phenotypes acquired by IS vs Tn insertions
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Consequences of transposon insertion
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The Tn5 transposon (5,8 kb) kanr bler strr
Tnp Tnp O I I O IS50L IS50R Transposase acts upon I and O in correct orientation Resistance to kana-, bleo- and streptomycin Cut-and-pase conservative transposition Random transposition Broadest host spectrum known
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Minitransposon engineering: the facts
Transposase acts on the outermost 19 bp I & O ends of the transposon I end O end 2. Transposase acts in cis even if present outside the transposon ends tnp tnp I O I O
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Minitransposon engineering
WT Tn5 O I I O Mini-transposon NotI NotI SfiI SfiI tnp* marker gene X I O
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pUC18Not & pUC18Sfi NotI or SfiI MCS pUC18 NotI or SfiI
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pUT 5.2 kb I O Select. marker Cloned DNA Tnp* ori R6K oriT bla SfiI
NotI NotI I Select. marker Cloned DNA O Tnp* ori R6K pUT 5.2 kb oriT bla
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l RK2 tra/mob R6K pir pUTs RK2 oriT R6K oriV
Conditional replication & suicide delivery of pUTs RK2 tra/mob R6K pir l pUTs RK2 oriT R6K oriV
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Donor strain lpir t Escherichia coli S171 lpir
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Transposition Ralstonia sp. Pseudomonas sp. Escherichia coli
Rhizobium sp. t lpir Escherichia coli
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Transposition Ralstonia sp. Pseudomonas sp. Rhizobium sp.
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Conjugation
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Tri-partite matings Recipient Helper Donor ColE1 oriV R6K tra/mob
oriT RK2 Helper Donor
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Tripartite mating
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Advantages of minitransposons as a system for heterologous DNA integration
- Stability. No rearrangements No transposition (transposase is lost) - Selection is not required - Several rounds of integration are feasible - Wide host spectrum
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Applications of mini-transposons
• Mutagenesis • Determination of essential genes • Gene expression studies
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Strategy for transposon mutagenesis of a bacterial strain
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Mapping insertions with pulse field EF
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Sequencing minitransposon insertions
NNNNNNNNNNN 3’ Primers I Km O 1st round PCR 2nd round
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Determination of essential genes 1- The negative approach
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Determination of essential genes 2- The positive approach
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Gene expression studies using minitransposons
• Engineering heterologous gene expression • Promoter probing • IVET (In vivo expression technologies)
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I R A B C R Bioindicadores basados en promotores catabólicos genes de
detoxificación Respuesta R A B C "natural" Antb R Informador Sistema informador Presentación de Emisión de luz o un epitopo fluorescencia Actividad enzimática
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Promoter probing • lacZ • luxAB • gfp • inu • gus • luc pUT O I
ori R6K Tnp* O reporter I Ab bla oriT • lacZ • luxAB • gfp • inu • gus • luc
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Reporter lacZ gene fusions
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Monitoring promoter activity
Inducer/ environmental signal Accumulation of beta-galactosidase
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Promoter probing Identification of postexponential promoters in P. Putida with Tn5 lacZ-tet minitransposons
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Model promoters with late and early response to inducers
LacZ fusions in minitransposons Pu Model promoters with late and early response to inducers Pm
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Phenotypes of Pm and Pu fused to lacZ-tet
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Transposons for promoter probing
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Growth phase-dependent promoters
probing Growth phase-dependent promoters
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Found promoters
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IVET In Vitro Expression Technology
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