Reversible, parallel and multitask cloning method and kit

#Field of the Invention

#The present invention is related to a reversible, parallel and/or multitask cloning method and kit, which improves the cloning of (preferably multiple) genetic element(s) in a nucleic acid construct such as a vector or the chromosome of a cell and the rapid and efficient selection of constructs with a correct integration of said genetic element(s), either in vitro or in vivo.

#More precisely, the present invention is related to integrated method and tools to construct recombinant DNA molecules (to be used as DNA vaccine or for gene therapy) without requiring the use of antibiotic(s) resistance gene(s) and without requiring the addition of one or more antibiotic(s) to the culture medium of cells submitted to this recombinant DNA method. The present invention allows to obtain the selection of recombinant host cell(s) transformed by a (exogenous) nucleic acid sequence of interest (extra-chromosomal vector containing the insert) and simultaneously stabilization (stable inheritance) of this (exogenous) nucleic acid sequence of interest into the transformed host cell(s) descendants (maintenance of the nucleic acid sequence of interest in the host cells population).

#FIG. 1 is an example of complex genetic construct obtained by parallel and multiple genetic events performed by the method of the present invention.

#FIGS. 2 to 5 are examples of reversible cloning and selecting method and kit according to the invention.

#FIG. 6 presents an example of a donor plasmid 110 comprising a nucleotide sequence of interest 11 flanked by recombination sites (111, 111′) and a receiving plasmid 13 (pKidCcdA) with a first nucleotide sequence 14 coding for a first poison protein (Kid=15) under the control of a promoter and flanked by recombination sites (16, 16′), and with a second nucleotide sequence 17 coding for an antidote protein (CcdA=18) to another second poison protein (CcdB=19) different from the first poison protein 15. These two plasmids 110 and 13 by the addition of a recombinase 120 will form a chimeric molecule 112 that is added to an E. Coli cell 12, comprising integrated in its genome a nucleotide sequence 113 encoding the poison protein CcdB 19.

#The present invention allows the making of complex genetic constructions through the use of (i) simultaneous and (ii) parallel events (the various recombinations and selection events present almost the same frequency). The “multitask” nature of the invention is defined as follows: for example, the invention allows to perform the insertion of genetic elements A and C, the deletion of genetic elements E and F, and the inversion of genetic elements B and D, some or all events (FIG. 1) being performed simultaneously in vitro (i.e., in the same tube) or in vivo (i.e., in the same organism). The final product of the above-mentioned events is a complex construct comprised of the genetic elements A, B, C, and D, all with the same orientation. The simultaneous selection of several genetic events (e.g., here, insertions, deletions, inversions, recombinations) is achieved through the use of a different selective marker (here poisons and antidotes genes for example) for each of the events. Filled black arrows represent promoters.

#Plasmid 1 is amplified in a strain resistant to poison 1. Plasmid 2 is amplified in a strain resistant to poisons 6 and 9. Plasmid 3 is selected in a strain:

#sensitive to poisons 1 and 6 (for the selection of the insertion of genetic elements A and C),