pMCY85

Protocol tips

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For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.	For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above.

Upstream tips
For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.
Protocol tips
For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above.

Publication protocol

"Carbenicillin was used in place of ampicillin in all cultures. All optical density at 600 nm (OD600) measurements were recorded on a Tecan Sunrise plate reader instrument (Männedorf, Switzerland) with 200 µL of culture in a standard 96-well plate.

For E expression experiments, E. coli cells were pre-cultured from single colonies in 5 mL of LB supplemented with appropriate antibiotic(s) at 25 °C for 16 h. Cells were then diluted into 10 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 50-mL flask and were cultured at 30 °C and 220 rpm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the cultures to a final concentration of 0.5 mM at the start of growth (t = −3 h) or once the cultures reached an OD600 of ~0.4 (t = 0) where indicated. Rhamnose was added to the cultures to final concentrations of 0.1, 0.2, or 0.5 mM once the cultures reached an OD600 of ~0.4.

For isolation of BMCs and purification of PduP-E, E. coli cells were pre-cultured from single colonies in 20 mL of LB supplemented with appropriate antibiotic(s) in a 250-mL flask at 25 °C for 16 h. Cells were then diluted into 200 mL of LB supplemented with appropriate antibiotic(s) to an initial OD600 of 0.03 in a 1-L flask. Cells were cultured at 30 °C and 220 rpm and harvested at 3 h post-rhamnose induction. All induction conditions with IPTG and rhamnose are the same as above."

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