What Are EZ-Tn5™ Transposomes™?
An EZ-Tn5™ Transposome™ (Figure 1) provides a rapid and easy method for generating a library of random gene knockouts in living bacteria. An EZ-Tn5 Transposome is a stable complex formed between an EZ-Tn5™ Transposon (a modified Tn5 transposon) and EZ-Tn5™ Transposase (a hyperactive Tn5 Transposase) in the absence of Mg 2+ (Figure 2).
EZ-Tn5 Transposomes are so stable that they can be introduced into living bacteria that can be transformed by electroporation. Once in the cell, the EZ-Tn5 Transposase is activated by Mg2+ in the host cell and the EZ-Tn5 Transposon DNA is randomly inserted into the host's genomic DNA. No other transposition system is so simple or versatile. Since there is no need for cell conjugation, suicide vectors, or specific host factors, EZ-Tn5 Transposomes are ideal for quickly and easily generating libraries of random gene knockouts, developing novel strains of bacteria or "tagging" bacteria for special applications.
| ||Figure 1. The 3-dimensional structure of a Tn5 Transposome. This graphic, based on x-ray crystallography data, was kindly provided by Ivan Rayment and William Reznikoff, University of Wisconsin-Madison. |
What EZ-Tn5 Transposomes are available and how are they used?
EZ-Tn5 Transposomes are available with either a kanamycin-resistance (Kanr) marker or a dihydrofolate reductase gene (DHFR; trimethoprim selection) marker.
EZ-Tn5™ <KAN-2>Tnp (Kanamycin selection)
EZ-Tn5™ <DHFR-1>Tnp Transposome (Trimethoprim selection)
Simply electroporate the EZ-Tn5 Transposome of choice into the electrocompetent bacteria of choice and allow the cells to recover. Transformed bacteria are selected on the appropriate medium containing kanamycin or trimethoprim. Surviving colonies contain an EZ-Tn5 Transposon randomly inserted into their genomic DNA. The number of transposition clones obtained is dependent on the transformation efficiency of the host cell.
The EZ-Tn5 Transposome system has been optimized so that on average just one EZ-Tn5 Transposon is inserted in the DNA of a cell. Transposon-insertion clones can be screened (Figure 3) using a phenotypic assay defined by the user, by genomic DNA sequencing, or PCR.
A Broad Host Range System
Researchers have been using EZ-Tn5 Transposomes for more than 15 years with more than 60 different species reported in the literature. Both Gram-negative and Gram-positive bacteria have been successfully transformed.
The most important requirement for success is preparation of electro-competent bacteria with the highest transformation efficiency possible.
Make Your Own EZ-Tn5 Transposome
Custom EZ-Tn5 Transposomes containing a user-defined Transposon DNA sequence, can be constructed using an EZ-Tn5™ Custom Transposome™ Construction Kit. Custom built Transposomes containing a gene, a regulatory sequence, a resistance marker, etc. (see Figure 4) have been reported.
| ||Figure 2. An EZ-Tn5™ Transposome™ is the stable complex formed by EZ-Tn5™ Transposon with EZ-Tn5™ Transposase in the absence of Mg2+. |
| ||Figure 3. An EZ-Tn5™ Transposome™ Complex can be electroporated into living cells where it randomly inserts the transposon component into the host's genomic DNA. The EZ-Tn5 Transposon insertion site can be analyzed by a variety of methods. |
| ||Figure 4. A custom EZ-Tn5™ Transposome™ was used to insert the bioluminescent reporter genes (luxCDABE) into E. coli. The "glowing" results are pictured here after addition of Hg+ which induces a mercury- dependent promoter. (Photograph courtesy of Bruce Applegate, Food Science Department, Purdue University). The "EZ::TN" name has been changed to "EZ-Tn5™". |
*The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited to EZ-Tn5™ Transposome™ complexes, is covered by U.S. Patent #6,159,736, #6,294,385 and related patent applications, exclusively licensed to Epicentre Technologies.