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