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 method for scarless stitching comprising
identifying at least three consecutive units of a genetic construct, wherein each of the at least three consecutive units is in a partial plasmid and is flanked by a 4-10 nucleotide upstream and downstream flanking sequence that forms 5′ cohesive ends when digested with a restriction enzyme, wherein the at least three consecutive units comprises a first unit, a second unit, and a third unit, wherein the first unit, the second unit, and the third unit each comprise one or more nif cluster genetic components,
digesting the first and second units with a first restriction enzyme and digesting the third unit with a second restriction enzyme to produce cohesive ends, and
ligating the cohesive ends to produce a plasmid.
2. The method of claim 1 , wherein the first unit is a regulatory unit.
3. The method of claim 2 , wherein the regulatory unit is a promoter unit.
4. The method of claim 1 , wherein the second unit is a Ribosomal binding site (RBS) unit, a coding DNA sequence (CDS) unit, or a combined RBS/CDS unit.
5. The method of claim 1 , wherein the third unit is a terminator unit.
6. The method of claim 1 , wherein the flanking sequences are 6 nucleotides in length.
7. The method of claim 1 , wherein the one or more nif cluster genetic components of the first unit, the second unit, the third unit, or any combination thereof is 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.
8. The method of claim 1 , wherein the one or more nif cluster genetic components of the first unit, the second unit, the third unit, or any combination thereof is derived from a nifUSVWZM operon from Klebsiella oxytoca.
9. The method of claim 1 , wherein the first unit comprises a transcription factor binding site.
10. The method of claim 1 , wherein the first unit, the second unit, or the third unit comprises at least one gene selected from the list consisting of: MA rufH, MfD, nifK, nifY, ntfE, rufN, ntfU, ntfS, nifV, ntfW, ntfZ, ntfM, nifF, MfB, and nifQ.
11. The method of claim 1 , wherein producing the plasmid by ligating the cohesive ends comprises removing a native regulatory element which is linked to the first unit, the second unit, or the third unit in the partial plasmid.
12. The method of claim 1 , wherein the plasmid comprises a synthetic regulatory element.
13. The method of claim 1 , wherein the plasmid comprises nif cluster genes which are in a different order relative to the order of the nif cluster genes in a corresponding native nif genetic cluster.
14. The method of claim 1 , wherein the plasmid comprises nif cluster genes which are reassembled in a different orientation relative to the orientation of the nif cluster genes in a corresponding native nif genetic cluster.
15. The method of claim 10 , wherein the plasmid comprises a promoter that has a different strength relative to the native promoter of the at least one gene.
16. The method of claim 10 , wherein the plasmid comprises a terminator that has a different strength relative to the native terminator of the at least one gene.
17. The method of claim 1 , wherein the plasmid comprises a controller.
18. The method of claim 1 , wherein the plasmid produced by the method comprises a refactored nif cluster.