Recombinant Cells and Organisms Having Persistent Nonstandard Amino Acid Dependence and Methods of Making Them

Abstract

Novel recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making novel recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

1 .- 17 . (canceled) 
     
     
         18 . A genetically modified cell comprising:
 an essential polypeptide producing or conferring susceptibility to one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the essential polypeptide at a particular position,   wherein the essential polypeptide is activated when the nonstandard amino acid is not present at the particular position, and   wherein the activated essential polypeptide confers susceptibility to the one or more toxins or toxic substances, which kills the recombinant cell or prevents or reduces proliferation of the recombinant cell.   
     
     
         19 . The genetically modified cell of  claim 18 , wherein one or both of folding and function of the essential polypeptide are disrupted when the essential polypeptide includes a nonstandard amino acid at the particular position. 
     
     
         20 . The genetically modified cell of  claim 18 , wherein the essential polypeptide produces or confers susceptibility to one or more toxins or toxic substances under specific environmental or growth conditions. 
     
     
         21 . The genetically modified cell of  claim 20 , in which the essential polypeptide is selected from the group consisting of one or any combination of SacB Tdk, GalK, ThyA, TolC, TetA, RpsL, and herpes simplex virus thymidine kinase. 
     
     
         22 . The genetically modified cell of  claim 18 , wherein multiple copies of a nucleic acid sequence encoding the essential polypeptide are present in the recombinant cell. 
     
     
         23 . The genetically modified cell of  claim 18 , wherein the recombinant cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell. 
     
     
         24 . A The genetically modified cell of  claim 18  further comprising:
 one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the one or more toxins or toxic substances at a particular position, 
 wherein the one or more toxins or toxic substances are activated when the nonstandard amino acid is not present at its particular position, and 
 wherein the activated toxins or toxic substances kill the recombinant cell or prevent or reduce proliferation of the recombinant cell. 
 
     
     
         25 . The genetically modified cell of  claim 24 , wherein the cell dies or experiences reduced or no proliferation if one or more of translation, folding or function is disrupted. 
     
     
         26 . The genetically modified cell of  claim 24 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell. 
     
     
         27 . A genetically modified cell comprising:
 1) an essential polypeptide encoded by an essential nucleic acid sequence, said essential polypeptide having a nonstandard amino acid substitution, wherein the absence of the nonstandard amino acid substitution disrupts one or any combination of translation, folding and function of the essential polypeptide; and   2) an essential polypeptide producing or conferring susceptibility to one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the essential polypeptide at a particular position,   wherein the essential polypeptide is activated when the nonstandard amino acid is not present at the particular position, and   wherein the activated essential polypeptide confers susceptibility to the one or more toxins or toxic substances, which kills the recombinant cell or prevents or reduces proliferation of the recombinant cell.   
     
     
         28 . The genetically modified cell of  claim 27 , wherein the cell dies or experiences reduced or no proliferation if one or more of translation, folding or function is disrupted. 
     
     
         29 . The genetically modified cell of  claim 27 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell. 
     
     
         30 . A genomically recoded organism comprising:
 1) a first essential polypeptide encoded by a first essential gene, said first essential polypeptide having a first nonstandard amino acid substitution, wherein the absence of the first nonstandard amino acid substitution disrupts one or both of folding and function of the first essential polypeptide; and   2) a second essential gene encoding a premature stop codon, wherein the presence of a second nonstandard amino suppresses the premature stop codon and allows translation of a second essential polypeptide.   
     
     
         31 . The genomically recoded organism of  claim 30 , comprising two or more stop codons. 
     
     
         32 . The genomically recoded organism of  claim 30 , further comprising one or more standard amino acid substitutions in the first essential polypeptide to accommodate the nonstandard amino acid substitution and maintain one or both of proper folding and proper function of the first essential polypeptide. 
     
     
         33 . The genomically recoded organism of  claim 30 , wherein the nonstandard amino acids are one or both of L-4,4′-Biphenylalanine and L-2-Naphthylalanine. 
     
     
         34 . The genomically recoded organism of  claim 30 , wherein the first essential polypeptide is the same as the second essential polypeptide. 
     
     
         35 . The genomically recoded organism of  claim 30 , wherein the first essential polypeptide is different than the second essential polypeptide. 
     
     
         36 . The genomically recoded organism of  claim 30 , wherein the nonstandard amino acid in the first essential polypeptide is the same as the nonstandard amino acid in the second essential polypeptide. 
     
     
         37 . The genomically recoded organism of  claim 30 , wherein the nonstandard amino acid in the first essential polypeptide is different than the nonstandard amino acid in the second essential polypeptide. 
     
     
         38 . The genomically recoded organism of  claim 30 , wherein the organism expresses a plurality of nonstandard amino acids. 
     
     
         39 . The genomically recoded organism of  claim 30 , wherein one or both of the first and second essential genes are selected from the group consisting of one or any combination of tyrS, alaS, pgk, metS, metG, adk, and holB. 
     
     
         40 . The genomically recoded organism of  claim 30 , wherein the organism is weakened or dies if folding of the first essential polypeptide is disrupted, if a function of the first essential polypeptide is disrupted, or if translation of the second essential polypeptide is terminated at the premature stop codon. 
     
     
         41 . The genomically recoded organism of  claim 30 , wherein the organism is selected from the group consisting of a yeast cell, a bacterium, a virion, a virosome, a virus-like particle, a plant cell, an insect cell, and a mammalian cell. 
     
     
         42 . The genomically recoded organism of  claim 30  further comprising:
 a toxin that is inactive when a nonstandard amino acid is present in the toxin at a particular position, wherein the toxin is activated when the nonstandard amino acid is not present at the particular position, and wherein the activated toxin kills the organism. 
 
     
     
         43 . The genomically recoded organism of  claim 42 , wherein the organism is weakened or dies if folding of the first essential polypeptide is disrupted, if a function of the first essential polypeptide is disrupted, or if translation of the second polypeptide is terminated at the premature stop codon. 
     
     
         44 . The genomically recoded organism of  claim 42 , wherein the organism is selected from the group consisting of a yeast cell, a bacterium, a virion, a virosome, a virus-like particle, a plant cell, an insect cell, and a mammalian cell. 
     
     
         45 . A method of making an NSAA-dependent genetically modified cell comprising:
 providing to the cell an essential polypeptide encoded by an essential nucleic acid sequence, said essential polypeptide having a nonstandard amino acid substitution at a particular position, wherein the absence of the nonstandard amino acid substitution at the particular position disrupts one or any combination of translation, folding and function of the essential polypeptide, and wherein the recombinant cell dies or experiences reduced or no proliferation if one or more of translation, folding or function of the essential polypeptide is disrupted.   
     
     
         46 . The method of  claim 45 , wherein the essential polypeptide further comprises one or more standard amino acid substitutions in the essential polypeptide to accommodate the nonstandard amino acid substitution and maintain one or both of proper folding and proper function of the essential polypeptide. 
     
     
         47 . The method of  claim 45 , wherein the essential polypeptide has been computationally redesigned so that the essential polypeptide can no longer fold properly, function properly or both, when the essential polypeptide expresses a standard amino acid at the particular position. 
     
     
         48 . The method of  claim 47 , wherein the redesign is performed by random mutagenesis and selection. 
     
     
         49 . The method of  claim 47 , wherein the redesign is performed by targeted mutagenesis and selection. 
     
     
         50 . The method of  claim 47 , wherein the redesign is performed computationally using Rosetta software. 
     
     
         51 . The method of  claim 45 , wherein the essential polypeptide is conditionally essential under specific environmental or growth conditions. 
     
     
         52 . The method of  claim 45 , wherein the conditionally essential polypeptide confers antibiotic resistance or is another selection marker. 
     
     
         53 . The method of  claim 45 , further comprising providing to the cell two or more essential polypeptides encoded by two or more essential nucleic acid sequences. 
     
     
         54 . The method of  claim 45 , wherein the cell expresses a plurality of nonstandard amino acids. 
     
     
         55 . The method of  claim 45 , wherein the essential nucleic acid sequence is an essential gene or encodes an essential polypeptide that is essential for survival, growth, or proliferation. 
     
     
         56 . The method of  claim 45 , wherein the cell is selected from the group consisting of prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell. 
     
     
         57 . The method of  claim 45 , further comprising providing to the cell one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the one or more toxins or toxic substances at a particular position,
 wherein the one or more toxins or toxic substances are activated when the nonstandard amino acid is not present at its particular position, and   wherein the activated toxins or toxic substances kill the recombinant cell or prevent or reduce proliferation of the recombinant cell.   
     
     
         58 . The method of  claim 57 , wherein one or both of folding and function of the one or more toxins or toxic substances are disrupted when the one or more toxins or toxic substances includes the nonstandard amino acid at the particular position. 
     
     
         59 . The method of  claim 57 , wherein the one or more toxins or toxic substances are selected from the group consisting of one or any combination of barnase, Ccdb, Hok, Fst, ParE, MazF, Kid, ToxN, RelE, Doc, HipA and Mvpt. 
     
     
         60 . The method of  claim 57 , wherein multiple copies of a nucleic acid sequence encoding the one or more toxins or toxic substances are provided to the cell. 
     
     
         61 . The method of  claim 57 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell. 
     
     
         62 . A method of conferring susceptibility to one or more toxins or toxic substance to a cell comprising:
 providing to the cell an essential polypeptide producing or conferring susceptibility to one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the essential polypeptide at a particular position,   wherein the essential polypeptide is activated when the nonstandard amino acid is not present at the particular position, and   wherein the activated essential polypeptide confers susceptibility to the one or more toxins or toxic substances, which kills the recombinant cell or prevents or reduces proliferation of the recombinant cell.   
     
     
         63 . The method of  claim 62 , wherein one or both of folding and function of the essential polypeptide are disrupted when the essential polypeptide includes a nonstandard amino acid at the particular position. 
     
     
         64 . The method of  claim 62 , wherein the essential polypeptide produces or confers susceptibility to one or more toxins or toxic substances under specific environmental or growth conditions. 
     
     
         65 . The method of  claim 62 , wherein the essential polypeptide is selected from the group consisting of one or any combination of SacB Tdk, GalK, ThyA, TolC, TetA, RpsL, and herpes simplex virus thymidine kinase. 
     
     
         66 . The method of  claim 62 , wherein multiple copies of a nucleic acid sequence encoding the essential polypeptide are provided to the cell. 
     
     
         67 . The method of  claim 62 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell. 
     
     
         68 . The method of  claim 62 , further comprising providing to the cell one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the one or more toxins or toxic substances at a particular position,
 wherein the one or more toxins or toxic substances are activated when the nonstandard amino acid is not present at its particular position, and   wherein the activated toxins or toxic substances kill the cell or prevent or reduce proliferation of the cell.   
     
     
         69 . The method of  claim 68 , wherein the cell dies or experiences reduced or no proliferation if one or more of translation, folding or function is disrupted. 
     
     
         70 . The method of  claim 68 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell. 
     
     
         71 . A method of making an NSAA-dependent genetically modified cell that is susceptible to one or more toxins or toxic substance comprising providing to the cell
 1) an essential polypeptide encoded by an essential nucleic acid sequence, said essential polypeptide having a nonstandard amino acid substitution, wherein the absence of the nonstandard amino acid substitution disrupts one or any combination of translation, folding and function of the essential polypeptide; and   2) an essential polypeptide producing or conferring susceptibility to one or more toxins or toxic substances that are inactive when a nonstandard amino acid is present in the essential polypeptide at a particular position,
 wherein the essential polypeptide is activated when the nonstandard amino acid is not present at the particular position, and 
 wherein the activated essential polypeptide confers susceptibility to the one or more toxins or toxic substances, which kills the recombinant cell or prevents or reduces proliferation of the recombinant cell. 
   
     
     
         72 . The method of  claim 71 , wherein the cell dies or experiences reduced or no proliferation if one or more of translation, folding or function is disrupted. 
     
     
         73 . The method of  claim 71 , wherein the cell is selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome, a virus-like particle, a plant cell, an animal cell, an insect cell and a mammalian cell.