Helpful Hints: The Transposome Strategy for Creating Knockouts in Bacteria,
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
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
- Analyze gene function by creating gene knockouts (insertional mutations)
in living cells without using suicide vectors, conjugation, or specific
- Sequence bacterial genes without cloning using primers (provided
in the kits) that are homologous to the ends of the inserted transposon.
- Create mutations in vivo using an R6Kγori containing
transposon and rescue clone the region of genomic DNA into which the
transposon has been inserted.
- 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.
- Goryshin, IY et al. (2000) Nature Biotechnol. 18,
- Hoffman, L and Jendrisak, J (1999) Epicentre Forum 6(3),
- Derbyshire, KM et al. (2000) Epicentre Forum 7(2),
- Guilhabert, MR et al. (2001) Epicentre Forum 8(2),
- Fernandes, PJ et al. (2001) Microbiology 147,
- Shi, H et al. (2002) Mol. Biochem. Parasitol. 121,
- Dorsey, CW et al. (2002) Epicentre Forum 9(3),
- Lin, J et al. (2002) Antimicrob. Agents Chemother. 46,
- Visit www.EpiBio.com/tnp_cite for an updated
- Butterfield, YSN et al. (2002) Nucleic Acids Res. 30,
* 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.
[top of page]