Team:SCUT-China A/Protocols

Cell culture

Bl21 cells were cultured 12 h at 37 °C with shaking in 50ml Centrifuge tube with 10ml LB medium containing the appropriate antibiotic. These cultures were used to inoculate 100 mL of LB medium containing the appropriate antibiotics in a 500 mL Erlenmeyer flask at an initial OD600 of 0.6-0.8. To induce the expression of the dsz genes under the Ptac or Plac or PT7 promoter, 1 mM IPTG was added to the medium. Cultures were incubated 12h at 16 °C.

Whole cell catalysis

Cells were harvested by centrifugation, washed twice with 0.9% PBS, and the cell pellet was resuspended  in 20 mM PBS buffer at an OD600 of 2.0 in a 100 mL Erlenmeyer flask. This resting cells preparation was used for the BDS assays, which were carried out in an orbital shaker at 30 °C and 200 rpm after adding 4S compounds (DBT). Samples were collected periodically and, unless otherwise specified, extracted with an equal volume of acetonitrile for HPLC analysis of substrate consumed and product(s) generated after centrifugation at 14000*g for 10 min. To check the extracellular location of some 4S-pathway intermediates, resting cells were centrifugedat 14000*g for 10 min and the supernatant was extracted with acetonitrile and analyzed by HPLC. Three/four different biological samples were used to calculate substrate consumption and intermediates/product formation in all experiments reported in this work.

Analytical methods

HPLC was employed to analyze the concentration of the 4S compounds (DBT, DBTO2,and HBP) employing a C18 column; the mobile phase was a mixture acetonitrile/water (70:30) at 0.7mL min"1 flow rate. Peaks were monitored at 280nm wave lengths. Calibrations were performed using highly purified standards of each compound

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South China University of tecnology

GuangZhou,China

Protocols

DszB in cell extracts

For the addition using cell extracts, the recombinant strain BL21-dszB cells were grown as detailed above (Cell culture), washed twice with 0.9% NaCl and resuspended in 50 mM HEPES buffer pH 8.0 at an OD600 of 20, and disrupted by passage through an ultrasonic fragmentation 2h. Cell debris were removed by centrifugation at 16,000 g for 20 min at 4 °C, and the resulting supernatant was used as cell extract. Addition of crude cell extracts containing DszB (0.5 mg protein/mL) will be added to our desulfurization system.