Directed evolution of synthetic gene cluster
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.
1. A DNA library comprising
a plurality of distinct refactored Nif clusters, wherein each distinct refactored Nif cluster comprises a refactored version of a same native Nif cluster, said native Nif cluster comprising at least two genes selected from the list consisting of: NifJ, NifH, NifD, NifK, NifY, NifE, NifN, NifU, NifS, NifV, NifW, NifZ, NifM, NifF, NifB, and NifQ; and wherein each refactored Nif cluster is organized into transcriptional units which are composed of a plurality of modular units, each modular unit being composed of one or more genetic components selected from the group consisting of regulatory elements, protein coding sequences, non-coding sequences, and spacers, wherein each refactored Nif cluster is designed to encode a plurality of proteins of a biosynthetic pathway and wherein each of the plurality of distinct refactored Nif clusters is distinct from said native Nif cluster by at least one of the following: one or more promoters in the refactored Nif cluster are different in strength or number relative to the native Nif cluster, one or more ribosome binding sites (rbs) that regulate the expression of proteins encoded by genes in the refactored Nif cluster are different in strength relative to the native Nif cluster, one or more terminators in the refactored Nif cluster are different in number relative to the native Nif cluster, the expression of the proteins is under the control of one or more synthetic regulatory elements, non-essential genes in the refactored Nif cluster have been removed relative to the native Nif cluster, the addition of DNA encoding a restriction enzyme site to one of the 5′ or 3′ termini of the refactored Nif cluster wherein the DNA encoding a restriction enzyme site is not found in the 5′ or 3′ termini, a different number of genetic components are present relative to the native Nif cluster, the order of genes in the refactored Nif cluster are different than the order of genes relative to the native Nif cluster, orientation of genes in the refactored Nif cluster are different than the orientation of genes relative to the native Nif cluster, and a different number of genes is present relative to the number of genes in the native Nif cluster.
2. The library of claim 1 , wherein native Nif cluster non-coding DNA has been removed from at least one of the transcriptional units.
3. The library of claim 1 , wherein native Nif cluster non-coding DNA has been removed from all of the transcriptional units.
4. The library of claim 1 , wherein each refactored Nif cluster includes 10-100 transcriptional units.
5. The library of claim 1 , wherein native Nif cluster regulatory sequences have been removed from all of the refactored Nif clusters.
6. The library of claim 5 , wherein the native Nif cluster regulatory sequences are transcription factor binding sites.
7. The library of claim 1 , wherein the regulatory elements are selected from the group consisting of promoters, ribosome binding sites, ribozymes, and transcription terminators.
8. The library of claim 1 , wherein the refactored Nif clusters are monocistronic.
9. The library of claim 1 , wherein the refactored Nif clusters are polycistronic.
10. The library of claim 1 , wherein the plurality of refactored Nif clusters produce functional compounds or encode gene products that produce functional compounds.
11. The library of claim 10 , wherein a functional compound produced is ammonia.
12. A method of producing a diverse genetic library, comprising:
producing a plurality of distinct refactored Nif clusters from a same native Nif cluster, said native Nif cluster comprising at least two genes selected from the list consisting of: NifJ, NifH, NifD, NifK, NifY, NifE, NifN, NifU, NifS, NifV, NifW, NifZ, NifM, NifF, NifB, and NifQ; by preparing a plurality of modular units from one or more genetic components using a scarless stitching assembly, assembling the modular units in a plurality of distinct patterns to produce the plurality of distinct refactored Nif clusters, wherein the plurality of distinct refactored Nif clusters form the diverse genetic library wherein the genetic components are selected from the group consisting of regulatory elements, Nif cluster protein coding sequences, non-coding sequences, and spacers, wherein each of the plurality of refactored Nif clusters is a variant of a same native Nif cluster, and wherein each of the plurality of refactored Nif clusters is a variant of each other refactored Nif cluster in the library, such that each refactored Nif cluster in the library has a different regulatory element from one another.
13. The library of claim 1 , wherein each of the refactored Nif clusters in the library has a different architecture from one another, wherein architecture is selected from gene order, and orientation.
14. The library of claim 1 , wherein each of the refactored Nif clusters in the library include one or more exogenous nucleic acids.
15. The library of claim 1 , wherein each distinct refactored Nif cluster comprises an inducible promoter.