Molecular cloning

    Progress in challenging structural biology projects, such as membrane protein structure determination, is tied to the possibility to screen many different variants for overexpression and biochemical stability to identify ones with superior biochemical properties. This strategy requires robust methods to generate large numbers of constructs for different expression systems.

    Due to the lack of a suitable alternative we have developed a novel high-throughput cloning technology termed FX cloning. FX cloning combines attractive features of established methods that were thus far not unified in one single method. FX cloning allows subcloning, the straightforward transfer of a sequence-verified open reading frame (ORF) to a variety of expression vectors, but it avoids the common but undesirable feature of significantly extending target ORFs with cloning-related sequences. It leaves a minimal seam of only a single amino acid to either side of the protein. Furthermore, FX cloning is highly efficient and economical in its use. The full procedure takes place in one pot and does not require intermediate purification steps. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. More information, including a web-based service for automated primer design can be found at the FX cloning website. Cloning and expression vectors for FX cloning can be obtained via our Addgene page.
    Next to FX cloning, we have developed a generic method for the high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The incentive for this were our poor efficiencies of DNA manipulations in L. lactis. The vector backbone exchange, or VBEx procedure allows efficient cloning in a native plasmid optimal for the specific expression host and devoid of alien elements. The VBEx strategy is fully compatible with FX cloning and other high-throughput cloning methods. Plasmids for the high-throughput cloning for expression in L. lactis can be obtained via our Addgene page.

    Relevant Publications

    Geertsma ER, Dutzler R. (2011)
    A versatile and efficient high-throughput cloning tool for structural biology.

    Biochemistry. 2011 Apr 19;50(15):3272-8

    Geertsma ER, Poolman B. (2007)
    High-throughput cloning and expression in recalcitrant bacteria.
    Nat Methods. 2007 Sep;4(9):705-7