DNA MOLECULES AND METHODS

摘要

A DNA molecule comprising: a modified Group II intron which does not express the intron-encoded reverse transcriptase but which contains a modified selectable marker gene in the reverse orientation relative to the modified Group II intron, wherein the selectable marker gene comprises a region encoding a selectable marker and a promoter operably linked to said region, which promoter is capable of causing expression of the selectable marker encoded by a single copy of the selectable marker gene in an amount sufficient for the selectable marker to alter the phenotype of a bacterial cell of the class Clostridia such that it can be distinguished from the bacterial cell of the class Clostridia lacking the selectable marker gene; and a promoter for transcription of the modified Group II intron, said promoter being operably linked to said modified Group II intron; and wherein the modified selectable marker gene contains a Group I intron positioned in the forward orientation relative to the modified Group II intron so as to disrupt expression of the selectable marker; and wherein the DNA molecule allows for removal of the Group I intron from the RNA transcript of the modified Group II intron to leave a region encoding the selectable marker and allows for the insertion of said RNA transcript (or a DNA copy thereof) at a site in a DNA molecule in a bacterial cell of the class Clostridia. A method of introducing a nucleic acid molecule into a site of a DNA molecule in a bacterial cell of the class Clostridia, the method comprising the steps of: (i) providing a bacterial cell of the class Clostridia with the DNA molecule as described above and a DNA molecule capable of expressing a Group II intron-encoded reverse transcriptase; and (ii) culturing the bacterial cell under conditions which allow for removal of the Group I intron from the RNA transcript of the modified Group II intron and the insertion of said RNA transcript containing the selectable marker gene (or a DNA copy thereof) into said site. Preferably, the bacterial cell of the class Clostridia is a Clostridium sp. The DNA molecules and methods are useful for making mutations in Clostridium spp.