Methods for Rule-based Genome Design

Abstract

Methods and systems for designing, testing, and validating genome designs based on rules or constraints or conditions or parameters or features and scoring are described herein. A computer-implemented method includes receiving data for a known genome and a list of alleles, identifying and removing occurrences of each allele in the known genome, determining a plurality of allele choices with which to replace occurrences in the known genome, generating a plurality of alternative gene sequences for a genome design based on the known genome, wherein each alternative gene sequence comprises a different allele choice, applying a plurality of rules or constraints or conditions or parameters or features to each alternative gene sequence by assigning a score for each rule or constraint or condition or parameter or feature in each alternative gene sequence, resulting in scores for the applied plurality of rules or constraints or conditions or parameters or features, scoring each alternative gene sequence based on a weighted combination of the scores for the plurality of rules or constraints or conditions or parameters or features, and selecting at least one alternative gene sequence as the genome design based on the scoring.

What is claimed is: 
     
         1 . A method comprising culturing viable bacterial cells in growth media, wherein the viable bacterial cells comprise a recoded genome, wherein the recoded genome comprises at least one particular sense codon at all instances within a gene in a corresponding template genome that is changed to an alternative codon, wherein the gene is a gene required to maintain a fitness of at least 80% as calculated by doubling time when compared to parental non-recoded bacterial cells, and wherein:
 (i) the recoded genome comprises at least one instance where the at least one particular sense codon is reassigned to at least one non-standard amino acid, and wherein a gene of the template genome that encodes a cognate tRNA to the at least one particular sense codon is mutated, silenced, inactivated or removed in the recoded genome; and   (ii) the recoded genome comprises at least one instance where a trinucleotide sequence corresponding to the sequence of the at least one particular sense codon that is changed to an alternative codon (A) is changed to a trinucleotide sequence corresponding to the sequence of the alternative codon and (B) is   (I) within a region containing a first gene and a second gene that overlap in the template genome, or   (II) within a non-coding motif that is an untranslated motif selected from the group consisting of a ribosome binding site motif, an mRNA secondary structure, an internal ribosome pausing site motif, a terminator, a promoter and combinations thereof, wherein the trinucleotide sequence corresponding to the sequence of the alternative codon that is within the non-coding motif preserves a structure or function of the non-coding motif, and   wherein the trinucleotide sequence corresponding to the sequence of the alternative codon and that is within (I) the region containing a first gene and a second gene that overlap in the template genome or (II) the non-coding motif is a trinucleotide sequence corresponding to the sequence of a synonymous codon or a non-synonymous codon with respect to the at least one particular sense codon.   
     
     
         2 . The method of  claim 1 , wherein the recoded genome comprises the at least one particular sense codon in a corresponding template genome that is changed to an alternative codon genome-wide. 
     
     
         3 . The method of  claim 1 , wherein the recoded genome comprises at least two particular sense codons in a corresponding template genome that are changed to an alternative codon. 
     
     
         4 . The method of  claim 1 , wherein the recoded genome comprises at least seven particular sense codons in the corresponding template genome that are changed to an alternative codon. 
     
     
         5 . The method of  claim 1 , wherein the at least one particular sense codon is selected from the group consisting of AGG, AGA, AGC, AGU, UUG, UUA, UCG and UCA. 
     
     
         6 . The method of  claim 1 , wherein the at least one particular sense codon is a combination of at least two particular sense codons selected from the group consisting of AGG, AGA, AGC, AGU, UUG, UUA, UCG and UCA. 
     
     
         7 . The method of  claim 1 , wherein the gene of the recoded genome in which all instances of at least one particular sense codon in a corresponding template genome is changed to an alternative codon is an essential gene. 
     
     
         8 . The method of  claim 1 , wherein aR UAG codons are removed from the recoded genome. 
     
     
         9 . The method of  claim 1 , wherein a gene encoding a release factor is removed from the recoded genome. 
     
     
         10 . The method of  claim 1 , wherein the at least one non-standard amino acid is incorporated into an endogenous polypeptide expressed by the viable bacterial cells. 
     
     
         11 . The method of  claim 1 , wherein the viable bacterial cells are a non-standard amino acid dependent version of a 4 codon gene. 
     
     
         12 . The method of  claim 1 , wherein the viable bacterial cells are a biocontained strain in which all UAG codons have been removed. 
     
     
         13 . The method of  claim 1 , wherein at least one gene in the recoded genome of the viable bacterial cells is modified such that the at least one non-standard amino acid is required for the bacterial cells to remain viable. 
     
     
         14 . The method of  claim 13 , wherein the at least one gene in the recoded genome of the viable bacterial cells is modified comprises adk and tyrS. 
     
     
         15 . The method of  claim 1 , wherein the viable bacterial cells are multi-virus resistant bacterial cells. 
     
     
         16 . The method of  claim 1 , wherein the viable bacterial cells express a recombinant protein comprising the at least one non-standard amino acid. 
     
     
         17 . The method of  claim 1 , wherein the method further comprises purifying a recombinant protein expressed by the viable bacterial cells and/or formulating a pharmaceutical composition comprising a recombinant protein expressed by the viable bacterial cells. 
     
     
         18 . The method of  claim 1 , wherein not all instances of the trinucleotide sequence corresponding to the sequence of the at least one particular sense codon within a non-coding motif of the recoded genome are changed to a trinucleotide sequence corresponding to the sequence of the alternative codon. 
     
     
         19 . The method of  claim 1 , wherein the recoded genome comprises all instances of the trinucleotide sequence corresponding to the sequence of the at least one particular sense codon within a non-coding motif being changed to a trinucleotide sequence corresponding to the sequence of the alternative codon. 
     
     
         20 . The method of  claim 1 , wherein an instance of the at least one particular sense codon that is changed to an alternative codon at all instances within a gene in a corresponding template genome is within a region containing a first gene and a second gene that overlap in the template genome, and wherein the instance of the at least one particular sense codon that is changed to an alternative codon at all instances within a gene in a corresponding template genome is within a non-coding motif of the first gene and a coding motif of the second gene. 
     
     
         21 . The method of  claim 1 , wherein an instance of the at least one particular sense codon that is changed to an alternative codon at all instances within a gene in a corresponding template genome is within a region containing a first gene and a second gene that overlap in the template genome, and wherein (a) in the recoded genome the first gene and the second gene are moved to alternate sites such that they do not overlap or (b) the recoded genome comprises the first gene and a copy of the second gene, wherein the first gene and the copy of the second gene do not overlap. 
     
     
         22 . The method of  claim 1 , wherein the gene is a gene required to maintain a fitness of at least 87% as calculated by doubling time when compared to parental non-recoded bacterial cells. 
     
     
         23 . The method of  claim 1 , wherein the gene is a gene required to maintain a fitness of at least 93% as calculated by doubling time when compared to parental non-recoded bacterial cells. 
     
     
         24 . Viable bacterial cells comprising a recoded genome, wherein the recoded genome comprises at least one particular sense codon at all instances within a gene in a corresponding template genome that is changed to an alternative codon, wherein the gene is a gene required to maintain a fitness of at least 80% as calculated by a doubling time when compared to parental non-recoded bacterial cells, wherein:
 (i) the recoded genome comprises at least one instance where the at least one particular sense codon is reassigned to at least one non-standard amino acid, and wherein a gene encoding a cognate tRNA to the at least one particular sense codon is removed from the recoded genome; and   (ii) the recoded genome comprises at least one instance where a trinucleotide sequence corresponding to the sequence of the at least one particular sense codon that is changed to an alternative codon (A) is changed to a trinucleotide sequence corresponding to the sequence of the alternative codon and (B) is within a non-coding motif that is an untranslated motif selected from the group consisting of a ribosome binding site motif, an mRNA secondary structure, an internal ribosome pausing site motif, a terminator, a promoter and combinations thereof, wherein the trinucleotide sequence corresponding to the sequence of the alternative codon that is within the non-coding motif preserves a structure or function of the non-coding motif, and wherein the trinucleotide sequence corresponding to the sequence of the alternative codon and that is within the non-coding motif is a trinucleotide sequence corresponding to sequence of a synonymous codon or a non-synonymous codon with respect to the at least one particular sense codon.   
     
     
         25 . The viable bacterial cells of  claim 24 , wherein the gene is a gene required to maintain a fitness of at least 87% as calculated by doubling time when compared to parental non-recoded bacterial cells. 
     
     
         26 . The viable bacterial cells of  claim 24 , wherein the gene is a gene required to maintain a fitness of at least 93% as calculated by doubling time when compared to parental non-recoded bacterial cells.