Pseudomonas aeruginosa PAO1 harboring expression plasmid*.
Sterile LB Miller.
Proper antibiotics corresponding to plasmid selection.
Unnatural amino acid for incorporation.
Bacterial incubator set for 37oC.
100 mM phosphate buffer (PB) (pH 7).
BugBuster 10X protein extraction reagent (Merck, Billerica, MA, USA).
Turbonuclease (Sigma-Aldrich, St. Louis, MO, USA).
lysozyme (Sigma-Aldrich, Rehovot, Israel).
Protease inhibitor (Merck, Darmstadt, Germany).
Room temperature shaker.
Cooled centrifuge at 10000 g.
Prepare LB Miller with proper antibiotics.
Grow initial culture of PAO1 harboring plasmid at 37°C for 24 hours.
Dilute the initial culture 1:100 for another 24 hours of growth at 37oC. In case of unnatural amino acid incorporation, supplement culture with a final concentration of 1 mM unnatural amino acid.
Following 24 hours, sediment 1 mL of culture and wash with 1 mL of 100 mM phosphate buffer (PB) (pH 7).
Sediment the cells once again and resuspend with 100 μL of lysis solution composed of 90% (v/v) PB, 10% (v/v) BugBuster 10X protein extraction reagent (Merck, Billerica, MA, USA), Turbonuclease (Sigma-Aldrich, St. Louis, MO, USA), lysozyme (Sigma-Aldrich, Rehovot, Israel), and Protease inhibitor (Merck, Darmstadt, Germany).
Incubate cells with lysis solution for 30 min at room temperature while shaking.
Once lysis is done, centrifuge at 4oC 10,000 g for 10 min.
Supernatant contains soluble protein fraction and is considered as lysate.
Lysate was used for downstream analyses in the form of click reaction, SDS-PAGE, fluorescent imaging, and Western blot.