Directed Evolution of the Transcriptional Regulator DntR: Isolation of Mutants with Improved DNT-Response
Figure 2
Overview of the directed evolution process.
Five libraries were created in subsequent order, as indicated by the numbers 1–5. Library 1–3 were created using epPCR with Mutazyme II polymerase. The library size was in the range of 1–7×107 transformants/library. Library 4 was created using a modified variant of StEP using Mutazyme II polymerase, resulting in recombination of clones selected from library 3 with additional point mutations introduced by Mutazyme II. The best responding clones selected from library 4 were then recombined with StEP, this time using Pfu Polymerase. The success of recombination was confirmed by sequencing of 8 clones from each of library 4 and 5. Library 1–4 was grown in LB, while Library 5 was grown in both LB and M9*, with selection performed on cells grown in M9*. Each library was sorted 2–4 times (sorting and regrowth prior to another library generation indicated by dashed arrow).