High growth capacity auxotrophic escherichia coli and methods of use

Abstract

This invention provides high growth capacity strains of auxotrophic Escherichia coli and methods for generating thereof. The high growth capacity strains express a complementing auxotrophic plasmid that allows the strain to grow in the absence of the auxotrophic amino acid. Also, provided herein is a method for preparing a bacterial cell extract of a high growth capacity strain of auxotrophic Escherichia coli for use in an in vitro protein expression.

1 .- 34 . (canceled) 
     
     
         35 . A bacterial cell extract prepared from a high-growth capacity, auxotrophic strain of  E. coli  cells, wherein
 (a) the  E. coli  cells comprise an inactivated gene necessary for the synthesis of glutamine;   (b) the  E. coli  cells comprise an auxotrophic selection plasmid having an expression cassette comprising a constitutive promoter operably linked to the glnA gene; and   (c) the doubling rate of a population of the  E. coli  cell is less than 60 minutes in a growth media comprising glutamine.   
     
     
         36 . The bacterial cell extract of  claim 35 , further comprising a template nucleic acid comprising a gene of interest operably linked to at least one promoter. 
     
     
         37 . The bacterial cell extract of  claim 36 , further comprising an RNA polymerase that recognizes the promoter(s) operably linked to the gene of interest. 
     
     
         38 . The bacterial cell extract of  claim 35 , further comprising ribosomes, ATP, amino acids, and tRNAs. 
     
     
         39 . The bacterial cell extract of  claim 38 , wherein the tRNA is a suppressor tRNA. 
     
     
         40 . The bacterial cell extract of  claim 35 , further comprising a chaperone protein. 
     
     
         41 . The bacterial cell extract of  claim 40 , wherein the chaperone protein is selected from the group consisting of DsbA, DsbB, DsbC, DsbD, FkpA, SlyD, and a combination thereof. 
     
     
         42 . The bacterial cell extract of  claim 35 , wherein the inactivated gene is glnA. 
     
     
         43 . A reaction mixture comprising a template nucleic acid comprising a gene of interest operably linked to at least one promoter, wherein the reaction mixture comprises the bacterial cell extract of  claim 35 . 
     
     
         44 . The reaction mixture of  claim 42 , further comprising an RNA polymerase that recognizes the promoter(s) operably linked to the gene of interest. 
     
     
         45 . The reaction mixture of  claim 42 , further comprising ribosomes, ATP, amino acids, and tRNAs. 
     
     
         46 . The reaction mixture of  claim 44 , wherein the tRNA is a suppressor tRNA. 
     
     
         47 . The reaction mixture of  claim 42 , further comprising a chaperone protein. 
     
     
         48 . The reaction mixture of  claim 47 , wherein the chaperone protein is selected from the group consisting of DsbA, DsbB, DsbC, DsbD, FkpA, SlyD, and a combination thereof. 
     
     
         49 . The reaction mixture of  claim 35 , wherein the inactivated gene is glnA. 
     
     
         50 . A method for producing a biologically active protein of interest, comprising:
 mixing the bacterial cell extract of  claim 35  with a template nucleic acid comprising a gene of interest operably linked to at least one promoter, amino acids, nucleotides, and an energy source under conditions sufficient to transcribe and/or translate the template nucleic acid into the protein of interest.   
     
     
         51 . The method of  claim 50 , wherein the template nucleic acid is mRNA or DNA. 
     
     
         52 . The method of  claim 51 , wherein the template nucleic acid is DNA comprising an RNA polymerase promoter, and the bacterial cell extract comprises an RNA polymerase. 
     
     
         53 . The method of  claim 50 , wherein the amino acids comprise natural or non-natural amino acids. 
     
     
         54 . The method of  claim 50 , wherein the protein of interest is an antibody or IgG.