Directed evolution of synthetic gene cluster
AssigneeMassachusetts Institute of Technology
InventorsDehua Zhao, Christopher A. Voigt, Michael Joseph Smanski
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Abstract
The invention relates to methods and products for generating diverse libraries of genetic material. The products include libraries and constructed nucleic acids as well as kits and databases and systems thereof.
Claims24
-46. (canceled)
A method of refactored gene cluster assembly, comprising:
- using scarless stitching assembly to connect a plurality of genetic components into nucleic acid modular units, wherein each genetic component comprises one or more nif cluster protein coding sequences;
- PCR amplification of the nucleic acid modular units to produce modular units having a sequence that will produce cohesive ends upon enzymatic digestion;
- insertion of the modular units having cohesive ends into plasmids using the cohesive ends to assemble the modular units in a manner that dictates orientation and relative position of the modular units within the plasmid to produce multiple plasmids having different modular units embedded therein;
- and
- combining the multiple plasmids to produce plasmids comprising a complete refactored gene cluster.
-53. (Canceled)
A library, comprising
- a plurality of modular units, wherein each modular unit comprises one or more promoters, one or more terminators, and one or more nif cluster protein coding sequences, and wherein each modular unit has a structure different than other modular units in the library,
- wherein the combination of genetic components within each modular unit is distinct from a modular unit found in a native nif cluster based on the number, the order, or the orientation of the one or more nif cluster protein coding sequences.
-66. (canceled)
A system comprising at least one processor and memory communicatively coupled to the at least one processor and storing computer-executable instructions that, when executed by the at least one processor, perform a method of identifying an optimal set of modular units for assembly to produce a functional refactored nif cluster, the method comprising:
- receiving experimental data obtained by analyzing a DNA library comprising a plurality of distinct refactored nif clusters, each refactored nif cluster comprising a plurality of modular units, the experimental data comprising a functionality value generated by analyzing functionality of modular units or subsets thereof of the DNA library in the context of the nif cluster;
- identifying a desired functional refactored nif cluster by comparing the functionality values of the modular units or subsets thereof included within a set of putative refactored nif clusters and comparing the functionality values of the resultant putative refactored nif clusters.
(canceled)
A peptide barcode library, comprising a modular unit composed of one or more genetic components selected from the group consisting of regulatory elements, protein coding sequences, non-coding sequences, and spacers, and including at least one nif cluster protein coding sequence, wherein a unique peptide barcode is genetically fused to each nif cluster protein coding sequence.
The method of 47, wherein the one or more nif cluster protein coding sequences are derived from a Klebsiella oxytoca nif cluster, an Azotobacter vinelandii nif cluster, an Anabaena variabilis nif cluster, a Methanococcus maripaludis nif cluster, a Bradyrhizobium japonicum nif cluster, a Nodularia spumigena nif cluster nif cluster, a Nostoc sp. nif cluster, a Paludibacter propionicigenes nif cluster, a Pelosinus fermentans nif cluster, a Sinorhizobium meliloti nif cluster, a Arcobacter sp. nif cluster, a Frankia sp. nif cluster, a Rhodobacter capsulatus nif cluster, a Nostoc azollae nif cluster, or a Cyanothece sp. nif cluster
Dependencies are inferred from the claim text.