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Transposomics™
In Vivo Transposomics™
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EZ-TN5TM

In Vivo TransposomicsTM

EZ-Tn5™ Custom Transposome™ Construction Kits

Applications
  • In vivo
  • mutagenesis or strain development of microorganisms.
  • Plasmid/gene rescue.
  • DNA sequencing.
  • Gene expression studies.

The EZ-Tn5™ Custom Transposome™ Construction Kits contains all the reagents needed to build and use an EZ-Tn5 transposon in either an in vivo or in vitro insertion reaction. Using standard cloning methods and the included pMOD-2 or pMOD-3 Transposon vectors, you can create custom transposons with almost any DNA. Prepare a transposon complex with the included EZ-Tn5 Tranposase for in vivo experiments, or mix the transposon with a suitable target DNA (plasmid or purified genomic DNA) for an in vitro insertion reaction. The EZ-Tn5 Custom Transposome Construction Kit is provided with sufficient reagents for 20 in vivo Transposome or 10 in vitro insertion reactions.

Benefits
  • Choice of two transposon construction vectors.
  • Broad host range with over 60 Gram-negative or Gram-positive species documented.
  • EZ-Tn5 Transposase and reaction buffers included for in vivo or in vitro transposition reactions.
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Figure 1. Overview of the procedure for the EZ-Tn5™ Custom Transposome™ Construction Kits.



EZ-Tn5™ Tnp and EZ-Tn5™ Tnp Transposome™ Kits
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Applications
  • Rapid generation of knock-out mutants in bacterial cells.
  • Knock-in of genes for bacterial strain development.
  • "Tagging" bacteria with visible genetic markers for environmental localization studies.
  • Direct sequencing of bacterial chromosomal DNA.

EZ-Tn5™ Transposome™ complexes are formed between an EZ-Tn5™ Transposon and EZ-Tn5™ Transposase, and provide a simple and reliable method for generating a library of random gene knockouts in vivo.* Just electroporate the EZ-Tn5 Transposome into any of a broad range of living bacterial cells and select for a marker encoded by the EZ-Tn5 Transposon (Fig. 1). Because there is no need for cell conjugation, suicide vectors, or specific host factors, EZ-Tn5 Transposomes are ideal for creating mutants in species that have poorly described genetic systems or lack adequate molecular tools.


Ready-to-use EZ-Tn5 Transposomes* are available containing either a kanamycin selectable marker () or dihydrofolate reductase gene () that can be selected on plates containing trimethoprim. These markers are readily expressed in many Gram-negative bacteria. You can also create your own EZ-Tn5 Transposome using one of the EZ-Tn5 pMOD™ Transposon Construction Vectors and EZ-Tn5 Transposase.

All EZ-Tn5 Transposons contain unique primer-binding sites at each end for bidirectional sequencing. Hence, a gene knockout can be sequenced directly using bacterial genomic DNA as template and the primers provided with each Transposome. Transposon insertions made using anEZ-Tn5 ori/KAN-2>Tnp Transposome Kit can be rescued and the flanking DNA sequenced.

EZ-Tn5 Transposome-mediated insertions have been made in many different microorganisms, including Acinetobactor, Campylobacter, Escherichia, Mycobacterium, Proteus, Pseudomonas, Saccharomyces, Salmonella, Trypanosoma, Xylella,and others. The number of transposition clones obtained is highly dependent on the transformation efficiency of the host cell (Table 1).

Benefits
  • Rapid mutagenesis procedure is simpler and easier to use than chemical mutagenesis.
  • More efficient than using mini-transposons with suicide plasmids.
  • Broad host range: over 60 species of Gram-negative and Gram-positive bacteria transposed so far.
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Figure 1. The EZ-Tn5™ Transposon insertion site in bacterial DNA can be sequenced directly using genomic DNA isolated using the MasterPure™ Complete DNA Purification Kit and primers homologous to the ends of the transposon.

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* The use of Transposome™ complexes for 
in vivoinsertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to Epicentre.



EZ-Tn5™ ori/KAN-2>Tnp Transposome™ Kit

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Applications
  • Rescue cloning of transposon-mutagenized microbial genes (Fig. 1).
  • Rescue of plasmids from non-E. coli bacteria.

Among the advantages of transposon mutagenesis is that the transposon serves as a marker that can be used to clone and sequence the region of genomic DNA that has been disrupted. Nothing makes this cloning process easier than creating mutations in vivo with the EZ-Tn5™ ori/KAN-2>Tnp Transposome™ Kit.* In addition to encoding a broad host-range kanamycin resistance gene, the transposon contains an E. coli conditional origin of replication (R6Kγori). The presence of this origin of replication enables the propagation or "rescue" of the region of genomic DNA, or plasmid, into which the transposon has been inserted.


First, electroporate the Transposome into electrocompetent cells of the highest possible transformation efficiency. Activation of the Transposome by Mg2+ in the cell results in the random insertion of the EZ-Tn5 ori/KAN-2> Transposon into the host's genomic DNA. Select transposition clones on kanamycin plates or by screening for the loss of gene function.

Benefits
  • Easily recover and propagate plasmids from diverse bacterial genera that will not normally replicate in E. coli.
  • Simple rescue cloning process of mutagenized genes speeds up structure/function studies and sequencing.
  • Random insertion of transposon DNA assures excellent coverage of entire bacterial chromosome.
  • Rescue clones can be sequenced bidirectionally using the primers provided that are homologous to the ends of the transposon.

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Figure 1. The process for rescue cloning of transposon insertion sites in genomic DNA using the EZ-Tn5™ ori/KAN-2>Tnp Transposome™ and TransforMax™ EC100D™ pir+ or TransforMax™ EC100D™ pir-116 Electrocompetent E. coli.

*The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to Epicentre.


EZ-Tn5™ Transposon Construction Vectors

Applications
  • Construction of custom EZ-Tn5™ Transposons.
  • EZ-Tn5 Transposons made with pMOD-3ori/MCS> and pMOD-5ori/MCS> can be used for a variety of rescue cloning applications.*

Epicentre offers five different EZ-Tn5™ pMOD™ Transposon Construction Vectors* for the preparation of custom EZ-Tn5 Transposons (Table 1). Each vector contains a multiple cloning site (MCS) flanked by the hyperactive 19-bp Mosaic Ends (ME, denoted by ) that are specifically and uniquely recognized by EZ-Tn5 Transposase. To prepare the transposon, clone any DNA sequence of interest (e.g., selectable marker, control element, reporter gene) into the MCS and then generate the transposon either by PCR amplification or restriction enzyme digestion. Any of the Transposon Construction Vectors can be used to generate an EZ-Tn5 Transposon, but they offer different features.


The Transposon Construction Vectors pMOD-2 and pMOD-3ori/MCS> are pUC-based vectors. They consist of ME sequences that flank an MCS in a vector with a colE1 origin of replication (Fig. 1). EZ-Tn5 pMOD-3ori/MCS> also contains an R6Kγori within the ME sequences, which is useful for a variety of rescue cloning applications.

Benefits
  • The MCS enables easy cloning of any DNA of interest.
  • Hyperactive 19-bp Mosaic End sequences flanking the MCS for high-efficiency transposition using EZ-Tn5 Transposase.
  • Unique primer-binding sites at each end of the transposon for bidirectional sequencing of the insertion site using the Forward and Reverse Sequencing Primers (available separately). No need to design your own primers.
  • Three options can be used to prepare an EZ-Tn5 Transposon–digestion with Pvu II, digestion with PshA I, or PCR amplification using the PCR primers provided with the vector.

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Figure 1 . Features of the EZ-Tn5™ pMOD™ Transposon Construction Vectors.

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Figure 2 (click to enlarge). EZ-Tn5™ Transposon Construction Vectors pMOD™-2 andpMOD™-3ori/MCS> replicate in standard E. coli strains using a colE1 origin of replication. Transposons made with the pMOD™-3ori/MCS> vector also have an R6Kγori within the transposon, for rescue cloning applications.

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*The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent No. 6,159,736 and related patents and patent applications, exclusively licensed to Epicentre.



TypeOne™ Restriction Inhibitor

Applications
  • Increase the transformation efficiency of unmodified DNA2 by blocking type I R-M systems, which are widespread in Eubacteria and Archaebacteria.3
  • Increase the insertion efficiency of EZ-Tn5™ Transposome™ complexes.2

DNA transformation can be difficult to achieve in many bacterial strains due to the presence of one or more restriction and modification (R-M) systems that cleave unmodified DNA that is recognized as "foreign." TypeOne™ Restriction Inhibitor* provides a powerful method for increasing transformation efficiencies in bacterial strains with type I R-M systems. Developed as a unique preparation of a phage protein called ocr,
1 TypeOne Inhibitor is readily electroporated into cells along with transforming DNA. In vivo, the protein acts as a molecular decoy that blocks the DNA-binding site of type I R-M enzymes and inhibits DNA cleavage.

Benefits
  • Easy to use: Simply add TypeOne Restriction Inhibitor to unmodified DNA or a Transposome and electroporate using standard methods.
  • Blocks all known families of type I R-M enzymes1 and can therefore be used to increase transformation efficiencies in a variety of bacterial cells.

Table 1. Effect of TypeOne™ Restriction Inhibitor on transformation efficiencies.
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References
  1. Walkinshaw, M.D. et al. (2002) Molec. Cell 9, 187.
  2. Hoff man, L. et al. (2002) Epicentre Forum 9(2), 8.
  3. Murray, N.E. et al. (2000) Microbial. Molec. Biol. Rev. 64, 412.

*Covered by issued and/or pending patents.
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