An Easy and Efficient Method for Engineering Large Synthetic Constructs into the Chromosome

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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.  




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


Patent Information:
For Information, Contact:
Laurie Tzodikov
Licensing Associates
Princeton University
Thomas Kuhlman
Edward Cox