Compositions and Methods for Controlling Copy Number for a Broad Range of Plasmids and Uses Thereof

Abstract

The present invention provides compositions and methods for controlling the copy number for a broad range of plasmids and uses thereof. Disclosed is a host cell for conditional control of copy number of a plasmid, which host cell comprises a poly(A) polymerase gene that is operably joined to a conditionally inducible promoter, and a method for cloning and stably maintaining a DNA sequence encoding a heterologous polypeptide in the host cell.

1 . An  E. coli  host cell for production of a foreign product, wherein the foreign product is selected from the group consisting of DNA, RNA and a polypeptide,
 wherein the foreign product is toxic, unstable, and/or inhibits propagation of the host cell when DNA that encodes said foreign product is present in the host cell in an R1-type or colE1-type plasmid at high copy number, wherein said host cell comprises a poly(A) polymerase gene that (a) is operably joined to a conditionally inducible promoter, (b) is located in the chromosome of said host cell, and (c) catalyzes polyadenylation of a copA or RNAI antisense RNA molecule;   wherein the host cell is obtained by (i) removing or inactivating a constitutively-expressed poly(A) polymerase gene and introducing said poly(A) polymerase gene that is operably joined to the conditionally-inducible promoter into the chromosome of the host cell or (ii) operably joining the conditionally-inducible promoter to a poly(A) polymerase gene that is located in the chromosome of the host cell.   
     
     
         2 . A method for cloning and stably maintaining a foreign product in an  E. coli  host cell, wherein the foreign product is selected from the group consisting of DNA, RNA and a polypeptide, wherein the foreign product is toxic, unstable, and/or inhibits propagation of the host cell when DNA that encodes the foreign product is present in the host cell at high copy number, the method comprising,
 A. providing:
 (1) an  E. coli  host cell for conditional control of copy number of an R1-type or colE1-type plasmid, wherein said host cell comprises a poly(A) polymerase gene that (a) is operably joined to a conditionally inducible promoter, (b) is located in the chromosome of said host cell, and (c) catalyzes polyadenylation of a copA or RNAI antisense RNA molecule; 
 (2) an R1-type or colE1-type plasmid; and 
 (3) a DNA sequence encoding a foreign product; 
   B. ligating the DNA sequence into the R1-type or colE1-type plasmid to generate a recombinant plasmid;   C. introducing the recombinant plasmid into the host cell; and   D. growing the host cell containing the recombinant plasmid under conditions wherein said conditionally inducible promoter that is operably joined to said poly(A) polymerase gene in said host cell is not induced, thereby cloning and stably maintaining the DNA sequence encoding the foreign product.   
     
     
         3 . A method for cloning, stably maintaining and generating a foreign product in an  E. coli  host cell, wherein the foreign product is selected from the group consisting of DNA, RNA and a polypeptide, wherein the foreign product is toxic, unstable, and/or inhibits propagation of the host cell when DNA that encodes the foreign product is present in the host cell at high copy number, the method comprising:
 A. providing:
 (1) an  E. coli  host cell for conditional control of copy number of an R1-type or colE1-type plasmid, wherein said host cell comprises a poly(A) polymerase gene that (a) is operably joined to a conditionally inducible promoter, (b) is located in the chromosome of said host cell, and (c) catalyzes polyadenylation of a copA or RNAI antisense RNA molecule; 
 (2) an R1-type or colE1-type plasmid; and 
 (3) a DNA sequence encoding a foreign product; 
   B. ligating the DNA sequence into the R1-type or colE1-type plasmid to generate a recombinant plasmid;   C. introducing the recombinant plasmid into the host cell;   D. growing the host cell containing the recombinant plasmid under conditions wherein said conditionally inducible promoter that is operably joined to said poly(A) polymerase gene in said host cell is not induced, thereby cloning and stably maintaining the DNA sequence encoding the foreign product; and   E. contacting the host cell containing the recombinant plasmid with an inducing agent that induces expression of the poly(A) polymerase gene to which the conditionally inducible promoter is joined, thereby increasing the copy number of the recombinant plasmid and generating the foreign product.