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Herramientas genéticas
en microbiología
ambiental III Mini-
transposones
Víctor de Lorenzo y Marc VallsCentro Nacional Biotecnología,
Madrid
Insertion sequences vs. transposons
Phenotypes acquired by IS vs Tninsertions
Consequences of transposon insertion
The Tn5 transposon (5,8 kb)
IS50L IS50R
kanr
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
TnpTnp
OIO I
bler strr
Minitransposon engineering: the facts
1. 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
I O
tnp
I O
tnp
Mini-transposon
Minitransposon engineering
O
marker
I
gene X
WT Tn5
tnp*
OIO I
NotINotI SfiI SfiI
pUC18Not & pUC18Sfi
pUC18MCS
NotI or SfiI
NotI or SfiI
pUT5.2 kb
ori R6KTnp*
OSelect. markerI Cloned DNA
NotI NotI
bla
oriT
SfiI SfiI
R6K oriV
R6K pir
Conditional replication & suicide delivery of pUTs
RK2 oriT
RK2 tra/mob
pUTs
Donor strain
Escherichia coli S171 pir
pirt
Escherichia coli
Ralstonia sp. Pseudomonas sp.Rhizobium sp.
pir
t
Transposition
Transposition
Ralstonia sp. Pseudomonas sp.Rhizobium sp.
ConjugationConjugation
Tri-partite matings
Donor
Recipient
Helper
tra/mob
ColE1
oriV R6K
oriT RK2
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
I OKm
1st round
PCR
2nd round
NNNNNNNNNNN 3’ Primers
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)
R
Antb R
genes de detoxificación
I
A B C
Informador
Respuesta "natural"
Sistema informador
Emisión de luz o fluorescencia
Presentación de un epitopo
Actividad enzimática
Bioindicadores basados en promotores catabólicos
Promoter probing
pUT
ori R6KTnp*
Oreporter I Ab
bla
oriT
• lacZ• luxAB• gfp• inu• gus• luc
Reporter lacZ gene fusions
Accumulation of beta-galactosidase
Monitoring promoter activity
Inducer/environmental
signal
Promoter probing
Identification of postexponential promoters in P.
Putida with Tn5 lacZ-tet minitransposons
Model promoters with late and early response to
inducers
Pu
Pm
LacZ fusions in minitransposons
Phenotypes of Pm and Pu fused to lacZ-tet
Transposons for promoter probing
Promoter probing
Growth phase-
dependent promoters
Found promoters
IVET
In
Vitro
Expression
Technology