Dna molecules and methods

Abstract

The present application discloses 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, wherein the marker gene comprises a Group I intron in forward orientation of causing expression in a bacteria cell of the class Clostridia and wherein the DNA molecule comprises sequences that allow for the insertion of the RNA transcript of the Group II intron in the chromosome of 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 is also provided. The DNA molecule and the method are useful for making mutations Clostridium app.

1 . 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 modified selectable marker gene comprises a region encoding a selectable marker and a promoter operably linked to said region, wherein the promoter causes expression of the selectable marker encoded by a single copy of the modified 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 modified 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.   
     
     
         2 - 39 . (canceled) 
     
     
         40 . The DNA molecule of  claim 1 , further comprising exons flanking the modified Group II intron, wherein the exons allow splicing of an RNA transcript of the modified Group II intron. 
     
     
         41 . The DNA molecule of  claim 40 , wherein the modified Group II intron further comprises targeting portions. 
     
     
         42 . The DNA molecule of  claim 41 , wherein the targeting portions guide insertion of the RNA transcript of the modified Group II intron into a site within a DNA molecule in the bacterial cell of the class Clostridia. 
     
     
         43 . The DNA molecule of  claim 42 , wherein the site is a selected site. 
     
     
         44 . The DNA molecule of  claim 43 , wherein the DNA molecule is a plasmid. 
     
     
         45 . The DNA molecule of  claim 44 , wherein the plasmid is an  Escherichia coli -Clostridia shuttle vector. 
     
     
         46 . The DNA molecule of  claim 45 , further comprising a region permitting conjugative transfer from  Escherichia coli  to a bacterial cell of the class Clostridia. 
     
     
         47 . The DNA molecule of  claim 1 , wherein the promoter operably linked to the region encoding the selectable marker is the promoter of the thl gene of  C. acetobutylicum,  the ptb gene of  C. acetobutylicum  or the adc gene of  C. acetobutylicum  or the promoter of the fdx gene of  C. perfringens  or the cpe gene of  C. perfringens.    
     
     
         48 . The DNA molecule of  claim 1 , wherein the selectable marker confers a growth advantage on a bacterial cell of the class Clostridia in which the selectable marker gene is expressed, compared to a bacterial cell of the class Clostridia lacking the selectable marker. 
     
     
         49 . The DNA molecule of  claim 48 , wherein the selectable marker confers erythromycin resistance or chloramphenicol resistance to the bacterial cell of the class Clostridia. 
     
     
         50 . The DNA molecule of  claim 1 , wherein the Group I intron is located within or upstream of the region encoding the selectable marker. 
     
     
         51 . The DNA molecule of  claim 1 , wherein the promoter operably linked to the modified Group II intron is an inducible promoter. 
     
     
         52 . The DNA molecule of  claim 51 , wherein the inducible promoter is inducible by isopropyl β-D-1-thiogalactopyranoside (“IPTG”) or xylose. 
     
     
         53 . The DNA molecule of  claim 51 , further comprising an open reading frame encoding a Group II intron-encoded reverse transcriptase operably linked to a promoter but not contained in the modified Group II intron. 
     
     
         54 . The DNA molecule of  claim 1 , wherein the bacterial cell of the class Clostridia is of the genus  Clostridium.    
     
     
         55 . The DNA molecule of  claim 54 , wherein the bacterial cell of the genus  Clostridium  is  C. thermocellum, C. acetobutylicum, C. difficile, C. botulinum, C. perfringens, C. beijerinckii, C. tetani, C. cellulyticum,  or  C. septicum.    
     
     
         56 . The DNA molecule of  claim 43 , wherein the selected site in the DNA molecule in the bacterial cell of the class Clostridia is located within a gene or within a portion of DNA which affects the expression of a gene. 
     
     
         57 . The DNA molecule of  claim 56 , wherein the site is located within the chromosomal DNA of the bacterial cell of the class Clostridia. 
     
     
         58 . A kit comprising (i) the DNA molecule of  claim 1  and (ii) a DNA molecule encoding a Group II intron-encoded reverse transcriptase. 
     
     
         59 . A method of splicing a nucleic acid molecule into a site in 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 of  claim 1  and a DNA molecule encoding a Group II intron-encoded reverse transcriptase; and   (ii) culturing the bacterial cell of the class Clostridia under conditions which allow for removal of the Group I intron from the RNA transcript of the DNA molecule of  claim 1  and insertion of the RNA transcript into said site.   
     
     
         60 . The method of  claim 59 , further comprising culturing the bacterial cell of the class Clostridia under conditions in which the selectable marker is expressed. 
     
     
         61 . The method of  claim 60 , further comprising selecting the bacterial cell of the class Clostridia based on an altered phenotype conferred by the selectable marker. 
     
     
         62 . The method of  claim 61 , further comprising the step of isolating a single clone of cells derived from the bacterial cell of the class Clostridia. 
     
     
         63 . The method of  claim 59 , wherein the bacterial cell of the class Clostridia is of the genus  Clostridium.    
     
     
         64 . The method of  claim 63 , wherein the bacterial cell of the genus  Clostridium  is  C. thermocellum, C. acetobutylicum, C. difficile, C. botulinum, C. perfringens, C. beijerinckii, C. tetani, C. cellulyticum,  or  C. septicum.    
     
     
         65 . The method of  claim 59 , wherein the site in the DNA molecule in the bacterial cell of the class Clostridia is located within a gene or within a portion of DNA which affects the expression of a gene. 
     
     
         66 . The method of  claim 65 , wherein the site is located within the chromosomal DNA of the bacterial cell of the class Clostridia. 
     
     
         67 . The method of  claim 59 , wherein the providing the bacterial cell of the class Clostridia with the DNA molecule comprises transducing or transferring the DNA molecule into the bacterial cell of the class Clostridia, or transconjugating the DNA molecule from a donor bacterial cell into the bacterial cell of the class Clostridia. 
     
     
         68 . A method of targeting a nucleic acid molecule to a selected site in a DNA molecule in a bacterial cell of the class Clostridia, the method comprising:
 (i) proiding a bacterial cell of the class Clostridia with the DNA molecule of  claim 41  and a DNA molecule encoding 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 DNA molecule of  claim 41  and insertion of said RNA transcript containing the selectable marker gene (or a DNA copy thereof) into said selected site.   
     
     
         69 . A method according to  claim 68 , wherein the selected site of the DNA molecule is a gene or a portion of DNA which affects the expression of a gene. 
     
     
         70 . A mutant bacterial cell of the class Clostridia obtained by the method of  claim 59 . 
     
     
         71 . A mutant bacterial cell of the class Clostridia obtained by the method of  claim 69 . 
     
     
         72 . A DNA molecule comprising a modified erythromycin-resistance gene containing a Group I intron which disrupts expression of the erythromycin resistance gene, wherein when the Group I intron is transcribed it is able to excise itself from the RNA transcript. 
     
     
         73 . A DNA molecule comprising a modified chloramphenicol-resistance gene containing a Group I intron which disrupts expression of the chloramphenicol resistance gene, wherein when the Group I intron is transcribed it is able to excise itself from the RNA transcript. 
     
     
         74 . A DNA molecule comprising a modified tetracycline-resistance gene containing a Group I intron which disrupts expression of the tetracycline resistance gene, wherein when the Group I intron is transcribed it is able to excise itself from the RNA transcript. 
     
     
         75 . A DNA molecule comprising a modified spectinomycin-resistance gene containing a Group I intron which disrupts expression of the spectinomycin resistance gene, wherein when the Group I intron is transcribed it is able to excise itself from the RNA transcript. 
     
     
         76 . The DNA molecule of  claim 72 , wherein the DNA molecule is a plasmid. 
     
     
         77 . A host cell comprising a DNA molecule according to  claim 76 . 
     
     
         78 . The DNA molecule of  claim 73 , wherein the DNA molecule is a plasmid. 
     
     
         79 . A host cell comprising a DNA molecule according to  claim 78 . 
     
     
         80 . The DNA molecule of  claim 74 , wherein the DNA molecule is a plasmid. 
     
     
         81 . A host cell comprising a DNA molecule according to  claim 80 . 
     
     
         82 . The DNA molecule of  claim 75 , wherein the DNA molecule is a plasmid. 
     
     
         83 . A host cell comprising a DNA molecule according to  claim 82 .