Univariant Extrinsic Initiator Control System for Microbes and an In Vitro Assembly of Large Recombinant DNA Molecules From Multiple Components

Abstract

The invention provides, inter alia, a nucleic acid (e.g. expression vector) that comprises at least a first coding sequence and a second coding sequence. Each conding sequence is under the control of an inducible promoter of defined strength. Different promoters can have different strengths. Each promoter is responsive to the same inducer. The invention also provides: methods of expressing coding regions, methods of making a product of a multi-enzyme pathway, and methods of optimizing the yield of a product of a multi-enzyme metabolic pathway using the nucleic acids provided by the invention. Also disclosed is a method of non-enzymatic gene cloning useful for practicing the invention.

What is claimed is: 
     
         1 . An expression vector, comprising:
 at least a first coding region and a second coding region;   the first coding region encoding at least a first gene product, the first coding region being operably linked to a first inducible promoter, the first inducible promoter being of a first strength and being responsive to an inducer; and   the second coding region encoding at least a second gene product, the second coding region being operably linked to a second inducible promoter, the second inducible promoter being of a second strength, different from the first strength, and being responsive to the inducer.   
     
     
         2 . The expression vector of  claim 1 , further including a third coding region encoding at least a third gene product, the third coding region being operably linked to a third inducible promoter, the third inducible promoter being of a third strength, different from the first strength and the second strength, and being responsive to the inducer. 
     
     
         3 . The vector of  claim 1 , wherein:
 the first coding region encodes at least a first enzyme, the first enzyme catalyzing a first reaction in a multi-step enzymatic pathway; and   the second coding region encodes at least a second enzyme, the second enzyme catalyzing a second reaction in the multi-step enzymatic pathway.   
     
     
         4 . The vector of  claim 3 , wherein the multi-step enzymatic pathway is the lycopene synthetic pathway or the amorphadiene synthetic pathway. 
     
     
         5 . The expression vector of  claim 1 , wherein the first and the second inducible promoters are each a derivative of a single RNA polymerase promoter. 
     
     
         6 . The expression vector of  claim 5 , wherein the derivative is an RNA polymerase promoter that includes a mutation in a region selected from a melting region or an initiation region. 
     
     
         7 . The expression vector of  claim 6 , wherein the RNA polymerase promoter is selected from a T7 RNA polymerase promoter, a T5 RNA polymerase promoter, a T3 RNA polymerase promoter, or an SP6 RNA polymerase promoter. 
     
     
         8 . A cell transfected with the vector of  claim 1 . 
     
     
         9 . (canceled) 
     
     
         10 . A kit, comprising at least two expression vectors,
 the first expression vector comprising a coding region encoding at least a first gene product, the coding region being operably linked to a first inducible promoter, the first inducible promoter being of a first strength and being responsive to an inducer; and   the second expression vector comprising a coding region encoding at least a second gene product, the coding region being operably linked to a second inducible promoter, the second inducible promoter being of a second strength, different from the first strength, and being responsive to the inducer.   
     
     
         11 . The kit of  claim 10 , wherein:
 the coding region of the first expression vector encodes at least a first enzyme, the first enzyme catalyzing a first reaction in a multi-step enzymatic pathway; and   the coding region of the second expression vector coding region encodes at least a second enzyme, the second enzyme catalyzing a second reaction in the multi-step enzymatic pathway.   
     
     
         12 . A method of expressing at least a first coding region and a second coding region in a cell, the method comprising:
 providing a cell comprising an expression vector of  claim 1  comprising at least the first coding region and the second coding region, wherein:
 the first coding region is operably linked to a first inducible promoter, the first inducible promoter being of a first strength and being responsive to an inducer, 
 the second coding region is operably linked to a second inducible promoter, the second inducible promoter being of a second strength, different from the first strength, and being responsive to the inducer; and 
   contacting the cell with the inducer, thereby expressing the first coding region and the second coding region.   
     
     
         13 . The method of  claim 12 , wherein
 the first coding region encodes at least a first enzyme, the first enzyme catalyzing a first reaction in a multi-step enzymatic pathway; and   the second coding region encodes at least a second enzyme, the second enzyme catalyzing a second reaction in the multi-step enzymatic pathway.   
     
     
         14 . The method of  claim 12 , wherein the expression vector further comprises a third coding region, the third coding region being operably linked to a third inducible promoter, the third inducible promoter being of a third strength, different from the first strength and the second strength, and being responsive to the inducer. 
     
     
         15 . A method of expressing at least a first coding region and a second coding region in a cell, the method comprising:
 providing a cell comprising at least a first expression vector comprising at least the first coding region encoding a first gene product, and at least a second expression vector comprising at least the second coding region encoding a second gene product, wherein:
 the first coding region is operably linked to a first inducible promoter, the first inducible promoter being of a first strength and being responsive to an inducer, 
 the second coding region is operably linked to a second inducible promoter, the second inducible promoter being of a second strength, different from the first strength, and being responsive to the inducer; and 
   contacting the cell with the inducer, thereby expressing the first coding region and the second coding region.   
     
     
         16 . A method of optimizing yield of a product of a multi-step enzymatic pathway in a host cell,
 the multi-step enzymatic pathway including at least a first reaction catalyzed by a first enzyme, and a second reaction catalyzed by the second enzyme, the method comprising:   determining optimal levels of expression of the first and the second enzymes;   determining the ratio of a strength of a first inducible promoter to a strength of a second inducible promoter, the ratio of the strengths corresponding to the optimal levels of expression of the first and the second enzymes, the first and the second promoters being responsive to the same inducer; and   constructing an expression vector of  claim 3  comprising:
 a first coding region encoding the first enzyme, the first coding region being operably linked to the first inducible promoter; and 
 a second coding region encoding the second enzyme, the second coding region being operably linked to the second inducible promoter. 
   
     
     
         17 . The method of  claim 16 , further including contacting the host cell with the inducer to induce expression of the first and the second enzymes. 
     
     
         18 . The method of  claim 16 , further including:
 determining an optimal level of expression of a third enzyme, the third enzyme catalyzing a third reaction in the multi-step enzymatic pathway;   determining the ratio of the strengths of the first inducible promoter to the second inducible promoter, to a third inducible promoter, the ratio of the strengths corresponding to the optimal levels of expression of the first enzyme, the second enzyme, and the third enzyme, the first, the second, and the third promoters being responsive to the same inducer; and   constructing an expression vector comprising:
 the first coding region encoding the first enzyme, the first coding region being operably linked to the first inducible promoter; 
 the second coding region encoding the second enzyme, the second coding region being operably linked to the second inducible promoter; and 
 a third coding region encoding the third enzyme, the third coding region being operably linked to the third inducible promoter. 
   
     
     
         19 . A method of gene cloning, comprising:
 contacting each of a vector and a set of inserts with a pair of first terminal primers, a pair of second terminal primers, and at least one pair of linking primers, wherein:
 the set of insets including at least a first and a second insert, the inserts in the set of inserts including at least a first coding region and a second coding region, 
 each of the first terminal primers includes a first region complementary to a region of the vector and a second region complementary to a region of a first insert, 
 each of the second terminal primers includes a first region complementary to a region of the vector and a second region complementary to a region of an insert different from the first insert, 
 each of the linking primers includes a first region complementary to a region of an insert in the set of inserts and a second region complementary to a region of a different insert in the set of inserts, and 
   
       wherein each primer includes at least one phosphorothioate internucleotide linkage;
 amplifying the vector and at least two inserts to produce a vector amplification product and at least two insert amplification products, each including at least one phosphorothioate internucleotide linkage; 
 non-enzymatically cleaving the vector amplification product and the at least two insert amplification products at the at least one phosphorothioate internucleotide linkage to produce complementary single-stranded overhangs; 
 annealing the vector amplification product and the at least two insert amplification products in the presence of a cation and thereby non-enzymatically assembling a transforming product; and 
 introducing the transforming product into a host cell. 
 
     
     
         20 . The method of  claim 19 , wherein the set of inserts includes at least one additional insert comprising at least one additional coding region, further including:
 contacting the at least one additional insert with a pair of linking primers;   amplifying the at least one additional insert to produce at least one additional insert amplification product;   non-enzymatically cleaving the at least one additional insert amplification product at the at least one phosphorothioate internucleotide linkage to produce complementary single-stranded overhangs;   annealing the vector amplification product, the at least two insert amplification products, and the at least one additional insert amplification product in the presence of a cation to non-enzymatically assemble the transforming product.   
     
     
         21 . The method of  claim 19 , wherein:
 the complementary single-stranded overhangs are at least 14 basepairs long;   the phosphorothioate internucleotide linkage is repeated every two or more nucleotides, and   annealing the vector amplification product and the at least two gene amplification products is performed in at least about 0.5 mM of a cation selected from Mg 2+ , Ca 2+ , Co 2+ , Cu 2+ , or a combination thereof.