pTip-QC2-gi_21221796

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Each of the plasmid vector was introduced into the host strain, R. erythropolis L-8827. The recombinant strains were precultured in LB liquid media containing 34 µg/ml chloramphenicol at 28 °C for overnight. 2 ml of the preculture was transferred to 20 ml of the same fresh media containing 0.5 µg/ml thiostrepton and cultured with 120 rpm agitation for 16 hours.

Protocol tips
Each of the plasmid vector was introduced into the host strain, R. erythropolis L-8827. The recombinant strains were precultured in LB liquid media containing 34 µg/ml chloramphenicol at 28 °C for overnight. 2 ml of the preculture was transferred to 20 ml of the same fresh media containing 0.5 µg/ml thiostrepton and cultured with 120 rpm agitation for 16 hours.

Publication protocol

A total of 204 genes (listed in Supplementary Data S1) from S. coelicolor A3(2) was cloned into the inducible expression vector pTip-QC212 at the NdeI restriction site (CATATG) in order to arrange the start codon at the same position. For genes whose start codon is other than ATG, the start codon was replaced with ATG. Each of the plasmid vector was introduced into the host strain, R. erythropolis L-8827. The recombinant strains were precultured in LB liquid media containing 34 µg/ml chloramphenicol at 28 °C for overnight. 2 ml of the preculture was transferred to 20 ml of the same fresh media containing 0.5 µg/ml thiostrepton and cultured with 120 rpm agitation for 16 hours. The cells were harvested and washed twice with 100 mM sodium phosphate buffer. The cell pellets were then resuspended with the same buffer containing 8 M urea and cell disruption was performed by glass beads using the Multi-beads shocker instrument (Yasui Kikai, Japan). The supernatant of the disrupted sample was used as the denatured crude cell extract.

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