Team:Tsinghua-E/Protocol

Protocols

(1) Strains:

BL21(DE3)(mut-bitter30(+))/pSC101wt-ori-mutD-sfGFP/pTrc99A-trpss-30-tetA

BL21(DE3)(mut-bitter32(+))/pSC101wt-ori-mutD-sfGFP/pTrc99A-trpss-32-tetA

Negative control：

BL21(DE3) pSC101wt-ori-sfGFP/pTrc99A-trpss-30-tetA

BL21(DE3) pSC101wt-ori-sfGFP/pTrc99A-trpss-32-tetA

Each evolution was conducted in three parallel with only one parallel of negative control.

(2) Genome mutation

Colonies were picked up from agar plate to culture in 5mL LB medium in a 15mL centrifuge tube with 50mg/L ampicillin and chloromycetin for overnight. The seed culture was added into the fresh 10mL LB medium with the same antibiotics in 50mL flask by volumn ratio of 1%. 1g/L arabinose was added during the component stage (OD600= 0.6-0.8). The culture was maintained at 37℃ for 24h.

(3) Evolution

The culture was centrifuged and resuspended by 2mL M9YE medium and cultured in 20mL M9YE medium with 50mg/L ampicillin, chloromycetin, 24mg/L IPTG and certain concentration of tetracycline (initial OD600 was about 0.02). OD600 of the culture was measured every two hour and transferred to the next M9YE medium culture with higher tetracycline concentration with OD600 within 0.3-0.35. For every round, 1mL culture was mixed with 1mL 50% glycerol and stored. One fraction of culture was cultured on agar plate to obtain single colony for further analysis. The remaining culture was continuouslymaintain in culture condition to stable stage for tryptophan HPLC analysis.
The evolution for each condition was conducted for three rounds. The concentration of tetracycline of the culture during evolution process was set as below.