Method of selection of allelic exchange mutants
AssigneeInstitut Pasteur
InventorsVladimir Pelicic, Jean-Marc Reyrat, Brigitte Gicquel
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Abstract
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.
Claims26
A process for replacing a nucleotide sequence in the genome of a mycobacterium, strain comprising the steps of:
- a) providing a vector containing SacB gene coding for levansucrase enzyme and a nucleotide sequence of interest;
- b) transforming the mycobacterium strain with said vector;
- c) selecting clones of the resulting transformed mycobacteria in which a nucleotide sequence in the genome has been replaced by the nucleotide sequence of interest by propagating said transformed clones in a culture medium supplemented with sucrose;
- and
- d) isolating a strain in which a nucleotide sequence in the genome has been replaced.
Recombinant vector pPR24 (C.N.C.M. No. I-1727).
Recombinant vector pPR25 (C.N.C.M. No. I-1728).
Recombinant vector pPR2 (C.N.C.M. No. I-1725).
Recombinant vector pPR26 (C.N.C.M. No. I-1729).
Recombinant vector pPR34 (C.N.C.M. No. I-1731).
A method for selecting mycobacteria transformed with the SacB gene comprising the steps of:
- a) providing a vector containing SacB gene coding for levansucrase enzyme;
- b) transforming a mycobacterium strain with said vector;
- c) cultivating the resulting transformed mycobacteria in a culture medium supplemented with sucrose;
- d) cultivating in parallel the same transformed mycobacteria in a culture medium without sucrose;
- and
- e) selecting clones sensitive to sucrose, indicating mycobacteria which have been transformed with the SacB gene.
A process for inserting a nucleotide sequence of interest in the genome of a slow growing mycobacterium strain comprising the steps of:
- a) providing a vector containing SacB gene coding for levansucrase enzyme and said nucleotide sequence of interest;
- b) transforming the slow growing bacterium with the vector of step a);
- c) selecting clones of the resulting transformed mycobacteria in which said nucleotide sequence of interest has been inserted by propagating said clones in a culture medium supplemented with sucrose;
- and
- d) isolating a strain in which said nucleotide sequence of interest has been inserted.
Recombinant vector pPR23 (C.N.C.M. No. I-1726).
Recombinant vector pPR27 (C.N.C.M. No. I-1730).
Dependencies are inferred from the claim text.