Lab grows macroscale, modular materials from bacteria RiceUniversity NatureComms
BL21 harboring plasmids pSMCAF015 and pSMCAF016, for expression of GFP and SpyCatcher-GFP, respectively, were inoculated in 25 mL of RM minimal media with 0.2% w/v glucose and 100 µg/mL ampicillin. After ~16 h of growth at 37 °C and 250 rpm, cells were used to inoculate 0.5 L of RM minimal media with 0.2% v/v glycerol, 100 µg/mL ampicillin and 0.0004% antifoam to a final OD~0.05. The cultures were allowed to grow at 37 °C until mid-log phase.
for 30 min, resuspended in lysis buffer and lysed using Avestin Emulsiflex C3 Homogenizer. The lysate was centrifuged at 12,000 ×for 1 h and the supernatant was collected for protein purification. The proteins were purified using Immobilized Metal Affinity Chromatography with a HisTrap FF column and buffers containing 50 mM Tris pH 8.0, 300 mM NaCl, 5% v/v glycerol, and 10–250 mM Imidazole.
for 10 min to immobilize the cells onto the silicon substrate. The silicon substrate was washed with 2 mL of sterile PYE to remove loosely-bound cells before being mounted to a metal puck and transferred to the AFM sample stage. In situ AFM was performed on an Asylum Cypher AFM using soft tapping mode. A fluid cell and two syringe pumps were assembled to control liquid flow and PYE medium was supplied to maintain cell viability during imaging.
BUD-ELM strain were cultured in standard or static conditions until they reached stationary phase . The supernatant of each culture was extracted and loaded onto a TGX Stain-Free™ gel . After running, the gel was transferred to a 0.2 μm nitrocellulose membrane and blocked for 1 h at room temperature with SuperBlock™ blocking buffer .
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