E.coli co-overexpression

Description

Technical info

Tilt Series Date: 2013-07-17

Data Taken By: Ariane Briegel

Species / Specimen: Escherichia coli

Strain: RP 437

Tilt Series Settings: Single Axis, tilt range: (-66.0°, 66.0°), step: 2°, constant angular increment, dosage: 160.0 eV/Ų, defocus: -3.0 μm, magnification: 41000x.

Microscope: Caltech Polara

Acquisition Software: UCSFTomo

Upload Method: reupload_downloaded

Processing Software Used: SIRT

Collaborators and Roles: strain sent from Heather Hodges (Kiessling lab), parent strain and plasmids published from the Parkinson lab

Purification / Growth Conditions / Treatment: Grew UU1607/pJC3/pPM25 overnight in 5mL TB + 3.8µl Cm + 5µl CarbDiluted back 1:50 into 200mL TB + 152µl Cm + 200µl Carb + 50µl 1M IPTG + 200µl Na-salicylateGrew ~2 hours to mid-log phaseSpun down 10' at 5,000 x gResuspended in 300µl lysis buffer + 0.1µl PMSF + 0.75 µl 1,10-phenanthrolineAdded 33.3µl (2mg/ml) fresh lysozyme solution (20mg/ml in 10mM Tris-HCl, pH 8.0)Incubated 45' at 37°CAdded 1.67µl 1M MgCl2, 0.2µl 1M CaCl2, 0.33µl DNase IIncubated 30' at 37°CSpun down 4000 g 5 min Resuspended in activity buffer (cold room) without glycerol

Attached Files

cooverexpressionatlas4micronmin20sanglesc.mrc, Tilt Series (Pixel Size 0.28 nm), 1.9 GB

cooverexpressionatlas4micronmin20sanglescSIRTrot.rec, Reconstruction (Pixel Size 0.0 nm), 11.4 GB