organism: Escherichia coli BW25113
description: Random mutagenesis was performed with N'-nitro-N-nitrosoguanidine (NTG), using as a parent strain the E. coli JCL16 (BW25113/F' [traD36, proAB , lacIq ZDM15]). Brieﬂy, exponential-phase cultures of JCL16 were concentrated two-fold by centrifugation and suspension in 0.1 M citrate buﬀer (pH 5.5) and exposed to N'-nitro-N-nitrosoguanidine(NTG) at ﬁnal concentration of 50 g/ml for 30 minutes at 37C to reach a percentage kill of approximately 50%. The cells were washed twice with 0.1 M phosphate buﬀer (pH 7.0) and grown in LB plus 4% glucose for two hours. The outgrown culture were then challenged in the presence of 14 g/L isobutanol which is highly toxic to the wildtype type strain JCL16, for 12 h and plated on LB agar plates containing 25 g/mL tetracycline. To select isobutanol tolerant mutants, isolated colonies were inoculated in 2 mL deep-96-well plates containing 300 L LB plus 8 g/L isobutanol per well and incubated for 24 h at 30C in a rotary shaker (250 rpm) (VWR). Bacterial growth was then determined by densitometry at 600 nm using a microplate reader (BioTek instruments Inc.)
attributes.tag: strain
attributes.value: JCL16 - BW25113/F′ [traD36, proAB+, lacIq ZΔM15]