This invention is a new method for generating highly diverse sets of nucleic acids in a rapid high throughput manner to create a multitude of different gene systems useful for bioengineering.
Though many cellular functions are encoded in gene clusters, they are challenging to manipulate due to their size and complexity. Large systems are difficult to engineer; the process is focused on combining parts at the primary DNA- sequence level making it tedious and slow. Currently, it is too expensive to simultaneously synthesize many alternative designs and native genetic systems are difficult to optimize. This invention is a strategy to perform directed evolution of synthetic gene clusters to rapidly achieve the designed functions and behaviors.
This technology uses a refactored nitrogen fixation (nif) gene cluster to build permutations that have radically reorganized genetics that could not be achieved starting from a wild-type cluster. Measured standard genetic parts and variants of those parts, such as promoters, ribosome binding sites and terminators can replace parts in a genetic cluster. Furthermore, configuration of the gene cluster is dramatically changed, including operon organization, division of cistrons, and order and direction of genes to create a huge diversity of genetics. Clusters of genes may be optimized to produce altered functionality resulting in different activity levels and profiles. For instance, the authors built 80 clusters that scrambled the architecture of all 16 essential nif genes (~2 Mb of DNA assembly) and found that the most functional variants had no aspects of the wild-type architecture conserved. This invention can rapidly generate synthetic gene clusters making it a useful tool for the field of bioengineering.
Rapid generation of diverse genetic clusters
Modified gene clusters for optimization or