Host-vector system for antibiotic-free CoIE1 plasmid propagation

Abstract

A host-vector system that uses the RNA-based copy number control mechanism of ColE1-type plasmids for regulating the expression of a marker gene allows for antibiotic-free selection of plasmids and is useful for production of plasmid DNA and recombinant proteins.

1 . A non-naturally occurring bacterial cell containing 
 i) a DNA sequence encoding a protein the expression of which is to be regulated, and, operably associated thereto,    ii) a DNA sequence encoding an RNA sequence that mimics an RNA II sequence, or parts thereof, and that is complementary to an RNA I sequence that is transcribable from a plasmid with a ColE1 origin of replication.    
     
     
         2 . The bacterial cell of  claim 1  containing said DNA sequences i) and ii) integrated in its genome.  
     
     
         3 . The bacterial cell of  claim 1  or  2 , wherein said DNA sequence i) is a DNA sequence that is foreign to said cell.  
     
     
         4 . The bacterial cell of  claim 3 , wherein said foreign DNA sequence i) encodes a protein that is lethal or toxic to said cell.  
     
     
         5 . The bacterial cell of  claim 4 , wherein said foreign DNA sequence i) is under the control of an inducible promoter.  
     
     
         6 . The bacterial cell of  claim 4 , wherein said foreign DNA sequence i) encodes a protein that is lethal or toxic to said cell per se or by generating a toxic substance.  
     
     
         7 . The bacterial cell of  claim 4 , wherein said foreign DNA sequence i) encodes a repressor protein that is lethal or toxic to said bacterial cell by repressing the transcription of a gene that is essential for growth of said cell.  
     
     
         8 . The bacterial cell of  claim 7 , wherein said essential gene is operably linked to a promoter which contains a DNA sequence that specifically bound by said repressor protein.  
     
     
         9 . The bacterial cell of  claim 8 , wherein said promoter linked to said essential gene is inducible.  
     
     
         10 . The bacterial cell of  claim 9 , wherein said inducible promoter is inducible independent of the inducible promoter of  claim 5 .  
     
     
         11 . The bacterial cell of  claim 1 , wherein said DNA sequence ii) is inserted between the ribosomal binding site and the start codon of said DNA sequence i).  
     
     
         12 . The bacterial cell of  claim 1 , wherein said DNA sequence i) and said DNA sequence ii) are linked such that they encode a fusion protein.  
     
     
         13 . The bacterial cell of  claim 1 , wherein said DNA sequence i) and said DNA sequence ii) are translationally coupled.  
     
     
         14 . The bacterial cell of  claim 1  that has the ability to replicate a plasmid with a ColE1 origin of replication.  
     
     
         15 . The bacterial cell of  claim 14 , wherein said cell is an Escherichia coli cell.  
     
     
         16 . A host-vector system comprising 
 a) a plasmid with a ColE1 origin of replication;    b) a bacterial host cell in which said plasmid a) can be replicated, containing 
 i) a DNA sequence encoding a protein the expression of which is to be regulated, and, operably associated thereto,  
 ii) a DNA sequence encoding an RNA sequence that mimics an RNA II sequence, or parts thereof, and that is complementary to an RNA I sequence transcribable from the plasmid a),  
 wherein said RNA sequence defined in ii), in the absence of the plasmid a), allows for expression of said protein and wherein, when said plasmid a) is present inside said host cell, the RNA I molecule transcribed from the plasmid hybridizes with said RNA sequence defined in ii), whereby expression of said protein is suppressed.  
   
     
     
         17 . The host-vector system of  claim 16 , wherein said bacterial host cell contains DNA sequences i) and ii) integrated into its genome.  
     
     
         18 . The host-vector system of  claim 16 , wherein said DNA sequence i) encodes a protein that is lethal or toxic to said bacterial cell and wherein said RNA sequence defined in ii), in the absence of the plasmid a), allows for expression of said lethal or toxic protein such that growth of said host cell is completely or partially inhibited and wherein, when said plasmid a) is present inside said host cell, the RNA I molecule transcribed from the plasmid hybridizes with said RNA sequence defined in ii), whereby expression of said lethal or toxic protein is suppressed such that said complete or partial growth inhibition is abrogated in plasmid-bearing cells.  
     
     
         19 . The host-vector system of  claim 16 , wherein said plasmid a) additionally contains a gene of interest.  
     
     
         20 . A method for producing plasmid DNA, comprising the steps of 
 i) transforming a population of bacterial host cells of  claim 4  with a plasmid that has a ColE1 origin of replication and contains a gene of interest that is not to be expressed from said plasmid in said bacterial host cell,    ii) growing said bacterial host cell population under conditions in which said lethal or toxic protein is expressible in the cells, whereby expression of said protein completely or partially inhibits growth of plasmid-free cells such that the plasmid-bearing cells outgrow the plasmid-free cells,    iii) harvesting plasmid-bearing cells, and    iv) isolating and purifying the plasmid DNA.    
     
     
         21 . A method for producing a protein of interest, comprising the steps of 
 i) transforming a population of bacterial host cells of  claim 4  with a plasmid that has a ColE1 origin of replication and contains a DNA sequence encoding a protein of interest under the control of a prokaryotic promoter that enables expression of said protein in said bacterial host cells,    ii) growing said bacterial host cell population under conditions in which said lethal or toxic protein is expressible in the cells, whereby expression of said protein completely or partially inhibits growth of plasmid-free cells such that the plasmid-bearing cells outgrow the plasmid-free cells,    iii) harvesting the protein of interest, and    iv) isolating and purifying it.