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EZ-Tn5 Transposome Kits

EZ-Tn5™ Tnp Transposome™, KAN-2 FP-1 Forward Primer, KAN-2 RP-1 Reverse Primer, and Sterile Water.

EZ-Tn5™ Tnp Transposome, KAN-2 FP-1 Forward Primer, R6KAN-2 RP-1 Reverse Primer, Sterile Water.

EZ-Tn5 Transposome Kits

Stably insert a selectable marker into bacterial strains for multiple applications, including mutagenesis and characterisation of gene function.
  • Generate random gene knockouts in living bacteria
  • Generate mutants with improved genetics or function across a broad host range of bacterial cell types
  • Characterise novel genes and gene functions
  • Identify genes involved in pathogenesis, toxicity, biofilm development
  • Unravel metabolic pathways
  • Identify essential genes and regulatory elements
  • 100s of citations for many different applications
Item ID MBTOOL-013
TBD
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Product information

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 (Figure 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.

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 localisation studies.
  • Direct sequencing of bacterial chromosomal DNA.

Ready-to-use EZ-Tn5 Transposomes* are available containing a kanamycin selectable marker (<KAN-2>). This marker is 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.

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.

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 an EZ-Tn5 <R6Kγ 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 Acinetobacter, 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.

Table 1. Examples of bacterial strains mutagenized using the EZ-Tn5 Tranposomes

Actinobacillus pleuropneumoniae
Agrobacterium tumefaciens
Bacillus subtilis
Bartonella henselae
Bdellovibrio bacterivorous
Campylobacter jejuni
Clavibacter michiganensis
subsp. sepedonicus
Corynebacterium diphtheriae
Enterobacter cloacae
Escherichia coli
Francisella tularensis
Haemophilus ducreyi
Moraxella catarrhalis
Mycobacterium avium
Mycobacterium bovis
(BCG)

Mycobacterium tuberculosis
Myxobacterium angiococcus
Neisseria gonorrhoeae
Pseudomonas putida
Pseudomonas syringae
Rhodococcus equi
Rickettsia prowazekii
Salmonella typhimurium
Serratia marcesens
Silicibacter pomeroyi
Spiroplasma citri
Streptococcus pyogenes
Xanthomonas campestris
Xylella fastidiosa
Zymomonas mobilis

Table 2. Number of Kan R transposon insertion clones produced from electroporation of 1 µl of EZ-Tn5 <KAN>Tnp Transposome

E. coli >10 5 Proteus vulgaris >103
Salmonella ty. >10 4 Mycobacterium smegmatis >10 2
Pseudomonas sp. >10 2

*Covered by issued and/or pending patents, exclusively licensed or assigned to Epicentre® (an Illumina® Company).

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