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

Insertion sequences vs. transposons

Phenotypes acquired by IS vs Tn insertions

Consequences of transposon insertion

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

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

Minitransposon engineering WT Tn5 O I I O Mini-transposon NotI NotI SfiI SfiI tnp* marker gene X I O

pUC18Not & pUC18Sfi NotI or SfiI MCS pUC18 NotI or SfiI

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

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

Donor strain  lpir t Escherichia coli S171 lpir

Transposition Ralstonia sp. Pseudomonas sp. Escherichia coli Rhizobium sp. t lpir Escherichia coli

Transposition Ralstonia sp. Pseudomonas sp. Rhizobium sp.

Conjugation

Tri-partite matings Recipient Helper Donor ColE1 oriV R6K tra/mob oriT RK2 Helper Donor

Tripartite mating

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

Applications of mini-transposons • Mutagenesis • Determination of essential genes • Gene expression studies

Strategy for transposon mutagenesis of a bacterial strain

Mapping insertions with pulse field EF

Sequencing minitransposon insertions NNNNNNNNNNN 3’ Primers I Km O 1st round PCR 2nd round

Determination of essential genes 1- The negative approach

Determination of essential genes 2- The positive approach

Gene expression studies using minitransposons • Engineering heterologous gene expression • Promoter probing • IVET (In vivo expression technologies)

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

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

Reporter lacZ gene fusions

Monitoring promoter activity Inducer/ environmental signal Accumulation of beta-galactosidase

Promoter probing Identification of postexponential promoters in P. Putida with Tn5 lacZ-tet minitransposons

Model promoters with late and early response to inducers LacZ fusions in minitransposons Pu Model promoters with late and early response to inducers Pm

Phenotypes of Pm and Pu fused to lacZ-tet

Transposons for promoter probing

Growth phase-dependent promoters probing Growth phase-dependent promoters

Found promoters

IVET In Vitro Expression Technology