Genetic Transformation of Bifidobacteria

摘要

The present invention concerns a method for genetically transforming a Bifidobacterium strain comprising a step of methylation of a shuttle vector in an E. coli or a Gram-positive bacterium strain by two type II DNA methyltransferases from a Bifidobacterium: a methyltransferase enzyme that methylates the adenine base at position 4 of the nucleotide sequence RTCAGG and a methyltransferase enzyme that methylates the cytosine base at position 4 of the nucleotide sequence GGWCC. The present invention also concerns genetic tools and culture media useful for carrying out said method.

主权项

1. A method for genetically transforming a Bifidobacterium strain, the method comprising:
i) transforming an Escherichia coli strain or a Gram-positive bacterium strain either with a recombinant vector DNA comprising a gene encoding a methyltransferase enzyme from a Bifidobacterium that methylates the adenine base at position 4 of the nucleotide sequence RTCAGG (BanLI methyltransferase) and a recombinant vector DNA comprising a gene encoding a methyltransferase enzyme from a Bifidobacterium that methylates the cytosine base at position 4 of the nucleotide sequence GGWCC (BanLII methyltransferase), or with a recombinant vector DNA comprising both a gene encoding a BanLI methyltransferase from a Bifidobacterium and a gene encoding a BanLII methyltransferase from a Bifidobacterium, wherein said recombinant vectors DNA are capable of replicating in said E. coli or Gram-positive bacterium strain, ii) transforming the E. coli or Gram-positive bacterium strain of step i) with a recombinant shuttle vector DNA comprising a DNA sequence of interest to introduce in a Bifidobacterium strain, wherein the said shuttle vector DNA is capable of replicating in the E. coli or the Gram-positive bacteria strain of step i) and in the Bifidobacterium strain to be targeted for genetic transformation, iii) cultivating the transformed E. coli or Gram-positive bacterium strain obtained in step ii), iv) extracting the shuttle vector DNA from the transformed E. coli or Gram-positive bacterium strain, v) transforming, preferably electrotransforming, a Bifidobacterium strain with the shuttle vector DNA obtained from step iv), vi) recovering the transformed Bifidobacterium strain of step v).