DNA plasmids with improved expression

Abstract

The present invention relates to the production and use of covalently closed circular (ccc) recombinant plasmids, and more particularly to vector modifications that improve expression of said DNA molecules in the target organism.

What is claimed is: 
     
       1. A method for heat inducible production of a Rep protein dependent replication origin plasmid vector, comprising:
 a. cloning the plasmid replication regulating Rep protein into an expression vector to create a P L -Rep protein expression cassette in which said Rep protein is expressed under the control of the P L  promoter and said P L  promoter further comprises an OL1 mutation in which repressor binding to OL1 is decreased or lost by mutation in OL1, said OL1 mutation comprising either a single base substitution or a single base deletion within the P L  promoter (−35 to −10) SEQ ID NO:10; 
 b. integrating said P L -Rep protein expression cassette into a host strain genome to create a P L -Rep protein host strain in which expression from the said P L -Rep protein expression cassette is repressed by a temperature sensitive lambda repressor expressed from the host strain genome; 
 c. transforming said P L -Rep protein host strain with said Rep protein dependent replication origin plasmid vector; 
 d. isolating the resultant transformed bacterial cells; 
 e. propagating said transformed bacterial cells at 25-32° C. to maintain the said Rep protein dependent plasmid vector at a basal copy number; and 
 f. inducing said transformed bacterial cells at 37-42° C. to increase copy number of said Rep protein dependent plasmid vector. 
 
     
     
       2. The method of  claim 1 , wherein said P L  promoter OL1 mutation comprising either a single base substitution or a single base deletion within the P L  promoter (−35 to −10) SEQ ID NO:10 is selected from the group consisting of a P L  promoter OL1-G (SEQ ID NO: 11), and a P L  promoter OL1-G to T (SEQ ID NO: 12). 
     
     
       3. The method of  claim 1 , wherein said Rep protein dependent replication origin is selected from the group consisting of a R6K replication origin, ColE2-P9 replication origin and ColE2 related replication origin. 
     
     
       4. The method of  claim 1 , wherein said plasmid replication regulating Rep protein comprises an R6K Rep protein mutation selected from the group consisting of P42L-P113S (SEQ ID NO: 13) and P42L-P106L-F107S (SEQ ID NO: 14). 
     
     
       5. The method of  claim 1 , wherein said plasmid replication regulating Rep protein comprises a ColE2 Rep protein selected from the group consisting of ColE2 Rep protein (SEQ ID NO: 15) and ColE2 Rep protein mutation G194D (SEQ ID NO: 16). 
     
     
       6. The method of  claim 1 , wherein said P L -Rep protein host strain further comprises a genomically expressed RNA-IN regulated selection marker. 
     
     
       7. The method of  claim 1 , wherein said Rep protein dependent plasmid vector for heat inducible production has a vector backbone with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41. 
     
     
       8. A method for heat inducible production of a Rep protein dependent plasmid vector, comprising:
 a. obtaining bacterial cells transformed with the Rep protein dependent plasmid vector, the bacterial cells comprising a P L  promoter and a Rep protein, wherein the Rep protein dependent plasmid vector comprises a replication origin, wherein the P L  promoter controls the expression of the Rep protein, wherein the P L  promoter comprises the nucleic acid sequence of SEQ ID NO:10, and wherein the P L  promoter comprises an OL1 mutation within the nucleic acid sequence of SEQ ID NO: 10, wherein the OL1 mutation comprises either a single base substitution or a single base deletion that decreases or prevents repressor biding to OL1;   b. incubating said bacterial cells at 25-32° C. to maintain the Rep protein dependent plasmid vector at a basal copy number; and   c. incubating said bacterial cells at 37-42° C. to increase copy number of said Rep protein dependent plasmid vector.    
     
     
       9. The method of claim 8, wherein said OL1 mutation within the nucleic acid sequence of SEQ ID NO:10 is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.  
     
     
       10. The method of claim 8, wherein the replication origin is selected from the group consisting of a R6K replication origin, ColE2-P9 replication origin and ColE2 related replication origin.  
     
     
       11. The method of claim 8, wherein the replication origin is a R6K replication origin, and wherein said Rep protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14.  
     
     
       12. The method of claim 8, wherein the replication origin is a ColE2-P9 replication origin, and wherein the Rep protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 16.  
     
     
       13. The method of claim 8, wherein said bacteria cells further comprises a genomically expressed RNA-IN regulated selection marker.  
     
     
       14. The method of claim 8, wherein said Rep protein dependent plasmid vector has a vector backbone with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.