Team:Northwestern/09 06

Notebook

Tuesday, September 6th

Tasks:

Jordan

    Figure 1: Troubleshooting summary

  • Ran periplasm fraction protocol on Cas9 culture in TetR
    • Centrifuged 25 mL suspension for 20 minutes at 4000 rpm and 4C
    • Discarded supernatant
    • Resuspended in 6.25 mL of ice-cold sucrose cell fractioning buffer 1
    • Incubated for 20 minutes, shook periodically
    • Centrifuged for 30 minutes at 4000 rpm and 4 degrees
    • Discarded supernatant
    • Resuspended in 1.56 mL ice cold buffer 2 with no detergents, transferred to eppendorf tube
    • Incubated for 20 minutes on ice
    • Centrifuged for 15 minutes at 11000 rpm on tabletop centrifuge
    • Saved supernatant, in fridge

Michelle

  • Gel extracted Cas9 for SS and Cas9 for iGEM
    • Nanodrop results:
      • Cas9 lnrz for SS: 9.0 ng/uL, 260/280: 1.8, 260/230: 0.44
      • Cas9 res.dig. for iGEM: 8.8 ng/uL, 260/280: 1.85, 260/230: 0.43

Paul

  • Got sequencing back for gRNA assembled construct
    • Results aligned under “Assembled Parts>pSB1T3…+gRNA”
    • Few point mutations on mRFP and Terminator-likely not too bad
  • Gibson assembly of YcdO (10 uL: *6:1 insert:bb, 1% DMSO, 4 hrs @50 C*)
    • 4.4 uL of Cas9 lnrz for SS (from 9/1/16 PCR, gel extracted 9/6/16- 9 ng/uL)
    • 0.5 uL of YcdO (from PCR products, 38 ng/uL)
    • 0.1 uL DMSO
    • 5 uL Gibson Assembly HiFi MasterMix
  • Met w/ Joe (w/ Tyler) about modeling
    • Detect potential off target cleavage sites of our gRNA in the gut microbiome
  • Restriction digest linear pSB1C3 and linear Cas9 (EcoRI, SpeI)

Sam

  • Sent Emma Nechamkin emails
    • Grew up two cultures (25 mL to start, ~22 mL now exist)
    • Both JC8031
      • One has pClyA-GFP-His6 Cam
        • Induced with 290 uL arabinose at about .5 absorbance (actual absorbance labelled)
      • One is normal
        • No antibiotic
    • Overnight in the 37°C shaking incubator
  • Found and watched antibiotics and CRISPR videos
  • Started adding graphics to pamphlet

Tyler

  • Mathematical modeling meeting w/Joe
  • Transformation of ligated products + Gibson SS (Ycdo)
    • 3µL Ligated Cas9 Product
    • 5µL Gibson SS Product
    • 1µL Transformation control (50pg mRFP control)
    • 2µL Gibson (+) control

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