EZ-Tn5™ [KAN-2]Tnp and EZ-Tn5™ [DHFR-1]Tnp Transposome™ Kits

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 (<KAN-2>) or dihydrofolate reductase gene (<DHFR-1>) 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 an EZ-Tn5 <R6Kγori/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.

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.

Table 1. Examples of bacterial strains mutagenized using the EZ-Tn5™ Tranposomes™
Actinobacillus pleuropneumoniae Agrobacterium tumefaciens Bacillus subtilis Bartonella henselae Bdellovibrio bacteriovorus 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 KanR transposon insertion clones produced from electroporation of 1 µl of EZ-Tn5™ <KAN>Tnp Transposome™
E. coli	>105	Proteus vulgaris	>103
Salmonella ty.	>104	Mycobacterium smegmatis	>102
Pseudomonas sp.	>102

*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.

Ordering

Catalog No.			Size

EZ-Tn5™ <KAN-2>Tnp Transposome™ Kit

TSM99K2

10 Reactions

EZ-Tn5™ <DHFR-1>Tnp Transposome™ Kit

TSM99D1

10 Reactions

Contents for all kits: Ready-to-use Transposome™ complex with either a KAN-2 or DHFR-1 marker, and forward and reverse primers for sequencing.