Synthetic gene clusters

A method for expressing one or more synthetic operons collectively associated with a function in a cell by replacing native regulation of a set of genes with synthetic regulation, the method comprising:

• providing coding sequences for a set of polypeptides encoded by genes collectively associated with a function;
• changing codon identity within at least one coding sequence by removing at least one regulatory sequence within the coding sequence, wherein removing the at least one regulatory sequence comprises replacement of native codons in the coding sequence with non-native synonymous codons and comprises selecting non-native codons having maximal distance from the native codons of the coding sequence;
• organizing the coding sequences into one or more synthetic operon(s);
• operably linking one or more heterologous transcriptional regulatory sequence to the operon(s), thereby controlling magnitude of gene expression from the operon(s);
• and
• expressing the one or more synthetic operon(s) in a cell under the control of a polypeptide that binds directly or indirectly to the heterologous transcriptional regulatory sequence;
• and
• detecting the magnitude of gene expression by computation, wherein the computation comprises a numerical optimization algorithm, and wherein the numerical optimization algorithm comprises the Nelder-Mead algorithm, the Newton's method, the quasi-Newton method, a conjugate gradient method, an interior point method, a gradient descent, a subgradient method, a ellipsoid method, the Frank-Wolfe method, an interpolation method and pattern search methods, or an ant colony model.

A method for expressing one or more synthetic operons collectively associated with a function in a cell by replacing native regulation of a set of genes with synthetic regulation, the method comprising:

• providing coding sequences for a set of polypeptides encoded by genes collectively associated with a function;
• changing codon identity within at least one coding sequence by removing at least one regulatory sequence within the coding sequence, wherein removing the at least one regulatory sequence comprises replacement of native codons in the coding sequence with non-native synonymous codons and comprises selecting non-native codons having maximal distance from the native codons of the coding sequence;
• organizing the coding sequences into one or more synthetic operon(s);
• operably linking one or more heterologous transcriptional regulatory sequence to the operon(s), thereby controlling magnitude of gene expression from the operon(s);
• operably linking a heterologous ribosomal binding site (RBS) to one or more coding sequence in the synthetic operon, wherein different RBSs are operably linked to different coding sequences, and wherein the RBSs regulate translation of the coding sequences in a ratio that is substantially similar to a ratio of translation from a native operon, and
• expressing the one or more synthetic operon(s) in a cell under the control of a polypeptide that binds directly or indirectly to the heterologous transcriptional regulatory sequence.

A method for expressing one or more synthetic operons collectively associated with a function in a cell by replacing native regulation of a set of genes with synthetic regulation, the method comprising:

• providing coding sequences for a set of polypeptides encoded by genes collectively associated with a function;
• changing codon identity within at least one coding sequence by removing at least one regulatory sequence within the coding sequence, wherein removing the at least one regulatory sequence comprises replacement of native codons in the coding sequence with non-native synonymous codons and comprises selecting non-native codons having maximal distance from the native codons of the coding sequence;
• organizing the coding sequences into one or more synthetic operon(s), wherein the synthetic operon comprises two functional sequences selected from the group consisting of a promoter, a ribosome binding site, a coding sequence, and a terminator and the method further comprises operably linking a buffer sequence between two functional sequences, and wherein the buffer sequence is selected from the group consisting of a random sequence, a UP-region of a promoter, an extended 5-UTR sequence, and a RNAase cleavage site;
• and
• expressing the one or more synthetic operon(s) in a cell under the control of a polypeptide that binds directly or indirectly to the heterologous transcriptional regulatory sequence.