Description:
Princeton
University Invention # 10-2603
Researchers in the
Department of Molecular Biology, Princeton University have developed an
efficient, easy to use, two-step system for the site-directed insertion of large
genetic constructs into arbitrary positions in the Escherichia coli chromosome.
This system can also be used for the integration of large DNA fragments into
bacterial artificial chromosomes (BACs) and plasmids. The system uses λ-Red
mediated recombineering accompanied by the introduction of double-stranded DNA
breaks in the chromosome and a donor plasmid bearing the desired insertion
fragment.
The method, as described
below, in contrast to existing recombineering or phage derived insertion
methods, allows for the insertion of very large fragments into any desired
location and in any orientation. The technology is also applicable to the
engineering of other bacteria and the development of tools, such as targeting
plasmids, applicable to transgenic animals such as mice.
In
the method, developed at Princeton, the cell is first transformed with a helper
plasmid, pTKRED, harboring genes encoding the λ-Red enzymes, I-SceI
endonuclease, and RecA. λ-Red enzymes expressed from the helper plasmid are used
to recombineer a small (1.3 kb) `landing pad¿, a tetracycline resistance gene
(tetA) flanked by I-SceI recognition sites and 25-bp landing pad regions, into
the desired location in the chromosome. After tetracycline selection for
successful landing pad integrants, the cell is transformed with a donor plasmid
carrying the desired insertion fragment; this fragment is excised by I-SceI and
incorporated into the landing pad via recombination at the landing pad regions.
In this manner, very large constructs can be inserted at any desired location
within the chromosome. After successful integration, the I-SceI recognition
sites in both the landing pad and the inserted fragment are eliminated, allowing
successive applications of this protocol without modification of the landing pad
regions. The entire procedure, from start to verified product, takes 1.5¿2
weeks. This method has proven to be very easy to use and highly successful,
allowing for the insertion of large (7 kb) fragments into several chromosomal
locations without a single failure.
References:
Kuhlman TE, Cox, EC,
Site-Specific Chromosomal Integration of Large Synthetic Constructs, Nucleic
Acids Research On line, 2009, 1-10.
Princeton is currently
seeking commercial partners for the further development and commercialization of
this opportunity.
For more
information on Princeton University invention # 10-2603 please
contact:
Laurie
Tzodikov
tzodikov@princeton.edu