Fig. 5: Accelerating adaptive laboratory evolution using OptoGPlad.
From: De novo designed protein guiding targeted protein degradation
a Schematic representation of OptoGPlad-assisted ALE. Under light exposure, continuous intracellular degradation of MutH increased the mutation rate and accelerated strain evolution. In the darkness, degradation of MutH ceased, allowing beneficial mutant strains to accumulate. b Binding interface selection (Above) and design models (Down) for GP complexes. Above: MutH surface showing hydrophobic residues (red) and Patchdock-selected binding sites (yellow). Right: Designed complexes of GPMutH-MutH, with binding sites (yellow) and targets (gray). c Left: WB analysis of intracellular MutH abundance after two hours of light-induced OptoGPlad expression. Right: WB analysis showing MutH accumulation four hours after transitioning to dark conditions. d Changes in the mutation rate of E. coli under the influence of OptoGPlad and PCA. Data represent mean ± s.d. from six biological replicates, each consisting of 20 parallel cultures. e Schematic representation of the evolution process and strain screening procedure. E. coli PCA06 was initially illuminated under blue light for 8 h and subsequently incubated for 16 h, representing round n. Following every n + 19 generation, cells with the highest fluorescence intensity were sorted by flow cytometry and used as chassis strains for the next round of evolution. f Propidium iodide (PI) staining was performed to assess the survival rate of strains at different stages of evolution in a medium containing 40 g/L PCA. The percentage of fluorescence intensity corresponded to the survival rate, where “−“ represented the live cell population and “+“ denoted the dead cell population. E. coli PCA05 strain cultured in fermentation medium without PCA was used as the control group. g PCA accumulation curves of E. coli PCA05 and E. coli PCA07 in the 5-L fermenter. All experiments are presented as mean ± s.d. from three biologically independent replicates. Source data are provided as a Source Data file. (a, e) are created in BioRender https://BioRender.com/ansn29h.
