1 Víctor de Lorenzo y Marc Valls Centro Nacional Biotecnología, MadridHerramientas genéticas en microbiología ambiental III Mini-transposones Víctor de Lorenzo y Marc Valls Centro Nacional Biotecnología, Madrid

2 Insertion sequences vs. transposons

3 Phenotypes acquired by IS vs Tn insertions

4 Consequences of transposon insertion

5 The Tn5 transposon (5,8 kb) kanr bler strrTnp 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

6 Minitransposon engineering: the factsTransposase 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

7 Minitransposon engineeringWT Tn5 O I I O Mini-transposon NotI NotI SfiI SfiI tnp* marker gene X I O

8 pUC18Not & pUC18Sfi NotI or SfiI MCS pUC18 NotI or SfiI

9 pUT 5.2 kb I O Select. marker Cloned DNA Tnp* ori R6K oriT bla SfiINotI NotI I Select. marker Cloned DNA O Tnp* ori R6K pUT 5.2 kb oriT bla

10 l RK2 tra/mob R6K pir pUTs RK2 oriT R6K oriVConditional replication & suicide delivery of pUTs RK2 tra/mob R6K pir l pUTs RK2 oriT R6K oriV

11 Donor strain  lpir t Escherichia coli S171 lpir

12 Transposition Ralstonia sp. Pseudomonas sp. Escherichia coliRhizobium sp. t lpir Escherichia coli

13 Transposition Ralstonia sp. Pseudomonas sp. Rhizobium sp.

14 Conjugation

15 Tri-partite matings Recipient Helper Donor ColE1 oriV R6K tra/moboriT RK2 Helper Donor

16 Tripartite mating

17 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

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

19 Strategy for transposon mutagenesis of a bacterial strain

20 Mapping insertions with pulse field EF

21 Sequencing minitransposon insertionsNNNNNNNNNNN 3’ Primers I Km O 1st round PCR 2nd round

22 Determination of essential genes 1- The negative approach

23 Determination of essential genes 2- The positive approach

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

25 I R A B C R Bioindicadores basados en promotores catabólicos genes dedetoxificación Respuesta R A B C "natural" Antb R Informador Sistema informador Presentación de Emisión de luz o un epitopo fluorescencia Actividad enzimática

26 Promoter probing • lacZ • luxAB • gfp • inu • gus • luc pUT O Iori R6K Tnp* O reporter I Ab bla oriT • lacZ • luxAB • gfp • inu • gus • luc

27 Reporter lacZ gene fusions

28 Monitoring promoter activityInducer/ environmental signal Accumulation of beta-galactosidase

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

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

31 Phenotypes of Pm and Pu fused to lacZ-tet

32 Transposons for promoter probing

33 Growth phase-dependent promotersprobing Growth phase-dependent promoters

34 Found promoters

35 IVET In Vitro Expression Technology

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