DOI: 10.22002/x84qf-35q36
title: Vibrio cholerae SAD2095

description: SAD2095 (T2SS and T4 competence pili) = rpoS*, pilA S81C, lacZ::lacIq, comEA-mCherry, ΔluxO, Ptac-tfoX, Ptac- recA, ∆TCP::ZeoR, ∆MSHA::CarbR, ∆CTX::KanR, ∆pilT::TmR Ankur: "For the taxonomy ID - unfortunately the strain we sent does not have a taxonomy ID. So, you can use the generic one for V. cholerae which is "666". The strain we are working with is related to N16961, but is certainly not the same strain, so not sure if you would want to indicate that somehow? Just for reference - the strain we sent is E7946 (although it is obviously heavily modified with a number of mutations). "
description: <strong>Tilt Series Date:</strong> 2019-07-14</p>
<strong>Data Taken By:</strong> Sara Weaver</p>
<strong>Species / Specimen:</strong> Vibrio cholerae</p>
<strong>Strain:</strong> SAD2095</p>
<strong>Tilt Series Settings:</strong>  Single Axis, tilt range: (-60.0°, 60.0°), step: 1.0°, constant angular increment, dosage: 120.0 eV/Å²,  defocus: -6.0 μm,  magnification: 26000x. </p>
<strong>Microscope:</strong> Caltech Titan Krios</p>
<strong>Acquisition Software:</strong> Serial EM</p>
<strong>Upload Method:</strong> pipeline</p>
<strong>Processing Software Used:</strong> Raptor</p>
<strong>Collaborators and Roles:</strong> Strain from Ankur B Dalia ankdalia@indiana.edu, PhD, Assistant Professor, Indiana University. Cells grown, data collected by Sara Weaver</p>
<strong>Purification / Growth Conditions / Treatment:</strong> SJW Microbiology Lab Notebook pages 7 and 8.  2019/03/05: Streaked stock on a plate. 2019/03/06: Grew up a single colony in 3 mL LB with shaking at 37˚C. 2019/03/07: The next day, subcultured 50uL of this overnight culture into 3mL of fresh LB + 100uM IPTG + 20mM MgCl2 + 10mM CaCl2 and grow at 30˚C shaking for ~5 hours.  This induced expression of the transcription factor TfoX to turn on the T2SS Type IV competence pilus.</p>
<strong>Sample Preparation:</strong> SJW Microbiology Lab Notebook pages 7 and 8. 2019/03/07 spun gold (100 µL 10 nm gold + 25 µL 5% BSA) at 15,000rpm for 21 minutes, removed supernatant. Resuspended gold beads in 100 µL liquid culture that was washed in 7 g/L instant ocean. Vitrobot RT and 95% humidity, 3µL of gold/sample slurry on carbon/copper Quantifoil Finder grids R2/2 (EmiTech glow discharge 50 mA 120 s) Blot force +6, blot time 4 s, no waiting time, no drain time. Plunged into ethane/propane mix.</p>
description: Files available via S3 at https://sdsc.osn.xsede.org/ini210004tommorrell/tomography_archive/swj2019-07-14-8/rawdata/</p>20190714_0309Box1grid1_SAD2095_target_TS18.mrc,  Tilt Series (Pixel Size 0.336 nm), 5.7 GB  
        
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        20190714_0309Box1grid1_SAD2095_target_TS18_part121_26.rec,  Reconstruction (Pixel Size 1.344 nm), 737.3 MB  
        
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        20190714_0309Box1grid1_SAD2095_target_TS18.mrc.mdoc,  63.9 kB  
        
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