Helpful Hints: The Transposome Strategy for Creating Knockouts in Bacteria, 114k PDF

Systems for In Vivo Transposomics™ Research

An EZ-Tn5™ Transposome™ complex is formed between an EZ-Tn5™ Transposon and EZ-Tn5™ Transposase (Figure 1). Although Transposomes are formed transiently during in vitro DNA insertion reactions, stable Transposomes can be prepared and isolated in the absence of Mg²⁺ (Figure 2). EZ-Tn5 Transposomes provide an efficient and reliable method for generating a library of random gene knockouts in vivo. 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 generating libraries of mutants in species that have poorly described genetic systems or lack adequate molecular tools.

EZ-Tn5 Transposomes are so stable that they can be electroporated into many living cells that can be transformed by electroporation.^1-9 Once in the cell, the EZ-Tn5 Transposome is activated by Mg²⁺ in the host's intracellular environment and the EZ-Tn5 Transposon component is randomly inserted into the host's genomic DNA. Cells containing the transposon are selected by plating on medium containing the antibiotic for which the EZ-Tn5 Transposon encodes resistance (Figure 3).

Figure 1. The 3-dimensional structure of a Tn5 Transposome.2 This graphic, based on x-ray crystallography data, was kindly provided by Ivan Rayment and William Reznikoff, University of Wisconsin-Madison.

A Broad Host Range System

EZ-Tn5 Transposomes have been used successfully by scientists working with a variety of different genera including Acinetobactor, Campylobacter, Escherichia, Mycobacterium, Proteus, Pseudomonas, Saccharomyces, Salmonella, Trypanosoma, Xylella, and more.^1-9 Various methods (e.g. Southern blot and sequence analysis) have verified that insertion mutants from these studies are both random and stable.

The number of transposition clones obtained is highly dependent on the transformation efficiency of the host cell. The higher the transformation efficiency of the cell, the more clones will be produced. Electroporation of an E. coli strain with a transformation efficiency for pUC19 DNA of >10⁹ cfu/µg typically results in >10⁵ independent insertion clones when 1 µl of EZ-Tn5 Transposome is used.

The presence of one or more type I restriction and modification systems may lower transposition efficiencies. Epicentre scientists have recently discovered that these systems can be blocked in vivo with the TypeOne™ Restriction Inhibitor.

	Figure 2. An EZ-Tn5™ Transposome™ is the stable complex formed by incubating an 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.

Sequencing or Rescue Cloning Your Gene Knockout Is Easy

The region of DNA into which the transposon is inserted can be sequenced directly using bacterial genomic DNA as template and primers homologous to the ends of the inserted EZ-Tn5 Transposon. You can also "rescue" clone the insertion site after using an EZ-Tn5 Transposome containing the R6Kγ origin of replication. Fragmented genomic DNA is simply self-ligated and the disrupted gene is propagated as a plasmid in a TransforMax™ E. coli strain expressing the pir gene product.

Use an Available EZ-Tn5 Transposome or Create Your Own

Ready-to-use EZ-Tn5 Transposomes are available containing either a kanamycin selectable marker (<KAN-2> or <R6Kγori/KAN-2>) or a marker selectable on trimethoprim (<DHFR-1>). Or make your own EZ-Tn5 Transposome containing virtually any DNA sequence of interest using one of the EZ-Tn5 pMOD Transposon Construction Vectors and EZ-Tn5 Transposase. For example, a custom EZ-Tn5 Transposon might include antibiotic resistant determinants for insertional mutagenesis, a reporter gene to facilitate studies of gene regulation and protein localization (Figure 4), or rare restriction enzyme sites for genome mapping.

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 was recently changed to "EZ-Tn5™" to distinguish EZ-Tn5 products from Epicentre's HyperMu™ transposon products.

HyperMu™ Transposome Complexes can also be used for in vivo insertions

Epicentre also offers HyperMu™ Transposome™ complexes to make insertions in living cells. HyperMu Transposomes are complexes between a HyperMu Transposon having R1 and R2 end sequences of bacteriophage Mu and HyperMu™ MuA Transposase, a hyperactive enzyme that retains the random insertion characteristics of MuA transposase¹⁰ but is at least 50 times more active in vitro than the MuA transposase available from other suppliers. Also, since EZ-Tn5 and HyperMu Transposases do not recognize the same end sequences for transposition, they can both be used when it is desirable to make more than one insertion. However, if you need to choose between Transposome systems, before beginning a project using a HyperMu Transposome, we recommend first comparing the results obtained in your organism using the HyperMu Transposome versus an EZ-Tn5 Transposome. In E. coli, EZ-Tn5 Transposomes typically generate 10-100 times more in vivo insertions than a HyperMu Transposome.

Applications of EZ-Tn5 and HyperMu Transposomes

1. Analyze gene function by creating gene knockouts (insertional mutations) in living cells without using suicide vectors, conjugation, or specific host factors.
2. Sequence bacterial genes without cloning using primers (provided in the kits) that are homologous to the ends of the inserted transposon.
3. Create mutations in vivo using an R6Kγori containing transposon and rescue clone the region of genomic DNA into which the transposon has been inserted.
4. Construct a custom EZ-Tn5 or HyperMu Transposome to introduce virtually any DNA sequence of interest into living cells. Use one of the EZ-Tn5 pMOD Transposon Construction Vectors to make EZ-Tn5 Transposons.

References

1. Goryshin, IY et al. (2000) Nature Biotechnol. 18, 97.
2. Hoffman, L and Jendrisak, J (1999) Epicentre Forum 6(3), 1.
3. Derbyshire, KM et al. (2000) Epicentre Forum 7(2), 1.
4. Guilhabert, MR et al. (2001) Epicentre Forum 8(2), 1.
5. Fernandes, PJ et al. (2001) Microbiology 147, 2529.
6. Shi, H et al. (2002) Mol. Biochem. Parasitol. 121, 141.
7. Dorsey, CW et al. (2002) Epicentre Forum 9(3), 14.
8. Lin, J et al. (2002) Antimicrob. Agents Chemother. 46, 2124.
9. Visit www.EpiBio.com/tnp_cite for an updated reference list.
10. Butterfield, YSN et al. (2002) Nucleic Acids Res. 30, 2460.

* The use of Transposome™ complexes for in vivo insertion of a transposon, including, but not limited to EZ-Tn5™ and HyperMu™ Transposome™ complexes, is covered by U.S. Patent #6,159,736, #6,294,385 and related patent applications, exclusively licensed to Epicentre Technologies.

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