Oct 05, 2025

Public workspaceStop Evolution

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld6q2og5b/v1
  • igemnthuth 1
  • 1iGEM_NTHU_Taiwan
  • iGEM NTHU
igemnthuth
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld6q2og5b/v1
Protocol Citation: igemnthuth 2025. Stop Evolution. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6q2og5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2025
Last Modified: October 05, 2025
Protocol Integer ID: 228983
Keywords: mutator dna polymerase, expression of the mutator dna polymerase, mutant strain, mutant strains for subsequent phenotypic analysis, mutation, evolution, rate evolution phase, strain, rhamnose, subsequent phenotypic analysis, dna, inducer
Abstract
Terminate the rhamnose-induced high-mutation-rate evolution phase by replacing the culture medium to remove the inducer, thereby shutting down expression of the mutator DNA polymerase in the OrthoRep system. This allows stabilization and selection of target mutant strains for subsequent phenotypic analysis.
Materials
  • Bacterial culture after directed evolution (containing rhamnose)
  • Fresh LB broth (without rhamnose)
  • Corresponding antibiotic(s)
  • PBS buffer
  • Centrifuge
  • Sterile conical tubes
Troubleshooting
Collecting the Culture
Take culture from the late stage of directed evolution (grown to OD₆₀₀ = 1.5–2.0).
Transfer into a 50 mL sterile centrifuge tube.
Harvesting Cells by Centrifugation
Centrifuge at 4500 × g, 4 °C, 5 min.
Carefully discard the supernatant completely.
Washing Step
Resuspend the cell pellet in 10 mL pre-chilled PBS.
Centrifuge again at 4500 × g, 4 °C, 5 min.
Completely discard the supernatant.
Resuspending Cells
Resuspend the pellet in 10 mL LB broth without rhamnose.
Add Antibiotic
Supplement the medium with the appropriate antibiotic.
Subsequent Culture
Incubate the culture at 37 °C, 220 rpm for 12 h.
Proceed with downstream experiments (e.g., sequencing, screening, fluorescence analysis).