Method of selection of allelic exchange mutants

Details Method of selection of allelic exchange mutants Method of selection of allelic exchange mutants

C - Chemistry, Metallurgy

12

N

C12N 15/65 (2006.01) C12N 1/21 (2006.01) C12N 9/10 (2006.01) C12N 15/54 (2006.01) C12N 15/74 (2006.01) C12N 15/90 (2006.01)

Patent

CA 2208315

A process for replacing a nucleotide sequence in the genome of a mycobacterium strain comprises the steps of: a) providing a vector containing SacB gene coding for levane saccharase enzyme and a nucleotide sequence of interest; b) transfecting the mycobacterium strain with the vector; c) selecting clones of the resulting transfected mycobacteria for replacement of the nucleotide sequence of interest by propagating the transfected clones in a culture medium supplemented with sucrose; and d) isolating the recombinant strain. The process is useful for positive selection of allelic exchange mutants, such as in Mycobacterium tuberculosis complex.

Procédé de remplacement d'une séquence nucléotidique dans le génome d'une souche de mycobactérie comprenant les étapes suivantes : a) fournir un vecteur renfermant le gène SacB encodant la saccharase et une séquence nucléotidique présentant un intérêt; b) transfecter la souche de mycobactérie avec le vecteur; c) sélectionner des clones des mycobactéries transfectées qui en résultent pour le remplacement de la séquence nucléotidique présentant un intérêt en propageant les clones transfectés dans un milieu de culture auquel on a jouté du saccharose; et d) isoler la souche recombinante. Le procédé est utile pour la sélection positive de mutants alléliques par échange de matériel génétique, comme dans le cas de Mycobacterium tuberculosis complex.

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