Oct 04, 2025
- igemnthuth 1
- 1iGEM_NTHU_Taiwan
- iGEM NTHU
Protocol Citation: igemnthuth 2025. Mutation. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21yeqg1y/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 04, 2025
Protocol Integer ID: 228967
Keywords: mutator dnap with rhamnose, mutation, mutator dnap, mutations for downstream analysis, mutagenesi, normal replication, rhamnose, evolution, evolution time
Abstract
Induce the mutator DNAP with rhamnose to drive mutations on the linear O-replicon; after the predetermined evolution time/generations, remove rhamnose to shut off mutagenesis and switch to “normal replication” to stabilize the mutations for downstream analysis.
Troubleshooting
Initial Culture (after O-replicon electroporation and initial screening)
Pick a single colony from the “IPTG & Kan screening” plate and inoculate into 15 mL LB + Kan (50 µg/mL).
Incubate overnight at 37 °C, 220 rpm for 12 h.
Start Mutagenesis
Transfer 5 mL of the overnight culture into 200 mL LB + Kan (50 µg/mL) in a 2 L baffled flask.
Grow until OD₆₀₀ ≈ 0.2.
Add 400 µL of 1 M rhamnose stock (final 2 mM) to immediately induce expression of the mutator DNAP.
Directed Evolution with Passaging
Total duration: 48 h, with passaging every 6 h.
Maintain incubation at 37 °C, 220 rpm; measure OD₆₀₀ at each 6 h interval.
Every 6 h: transfer 100 µL of the current culture into 10 mL fresh LB + Kan (50 µg/mL) + rhamnose (2 mM; add 20 µL of 1 M stock per 10 mL). Continue incubation at 37 °C, 220 rpm.
Sample preservation at each time point (recommended):
- 1 mL for glycerol stock (final 20%, stored at −80 °C).
- 1–2 mL saved separately for downstream PCR/sequencing.
- Continue this routine for a total of 48 h.
Stop Mutagenesis / Start Normal Replication
At the end of 48 h, when culture OD₆₀₀ reaches ~1.5–2.0, collect samples into 50 mL sterile centrifuge tubes.
Centrifuge at 4500 × g, 4 °C, 5 min; carefully discard the supernatant.
Wash: resuspend pellet in 10 mL ice-cold PBS → centrifuge 4500 × g, 4 °C, 5 min → discard supernatant.
Resuspend in 10 mL LB + Kan (50 µg/mL) without rhamnose.
Stabilization culture: incubate at 37 °C, 220 rpm, for 12 h (normal replication phase to shut down mutagenesis and stabilize genotype).