Team:Northwestern/09 01

Notebook

Thursday, September 1st

Transformation Results:

The gRNA constructs are assembling; Cas9 + YcdO once again failed to assemble.

Figure 1: mRFP in pSB1T3 transformation

Figure 2: mRFP in pSB1T3 transformation 2

Figure 3: Gibson negative control (no insert)

Figure 4: Gibson mix positive control

Figure 5: gRNA Gibson retransformation

Figure 6: Transformation positive control

Figure 7: YcdO + Cas9 Gibson

Tasks:

Jordan

  • Imaged periplasm fractions and ClyA-GFP with EVOS

    Figure 1: Whole cell ClyA-GFP

Michelle

  • Transferred TetR-Cas9 induced cultures to 10 mL tubes
  • Miniprepped GFP 1–3 in storage vectors
    • GFP1 colony A: 77.7 ng/uL, 260/280: 1.90, 260/230: 2.18
    • GFP1 colony B: 223.0 ng/uL, 260/280: 1.76, 260/230: 1.25
    • GFP2 colony A: 65.2 nh/uL, 260/280: 1.92, 260/230: 2.32
    • GFP2 colony B: 132.1 ng/uL, 260/280: 1.89, 260/230: 2.26
    • GFP3 colony A: 19.3 ng/uL, 260/280: 1.90, 260/230: 1.50
    • GFP3 colony B: 19.0 ng/uL, 260/280: 1.76, 260/230: 0.71
  • Grew 5mL overnight culture (x3) of gRNA from retransformed plate 5 mL LB + 5uL 1000X Tet
    • Miniprep tomorrow for electroporation cotransformation next week
  • Grew 5mL overnight culture (x2) of Cas9 from glycerol stock 5 mL LB + 5uL 1000X Cam
    • Miniprep tomorrow for electroporation cotransformation next week

Sam

  • Prepped questions for Dr. Scheetz
  • Looked into Australia protocol
  • Interviewed Dr. Scheetz

Sara

  • Transformed MIT plasmids

Tasfia

  • Made sequencing rxn mixtures for (~500 ng) gRNA-mRFP Gibson product based on Paul’s notes from 7.26.16 using the following primers
    • VR (Biobrick Rev)
    • Cas9-Seq4 (Fwd)
    • VF (Biobrick Fwd)
  • Gibson Reaction for Ycdo into “Cas9-Lrz-SS 1:3” linearized on 8.29.16
  • New conditions for Gibson:
    • Ran 5:1 molar ratio of insert-to-backbone, 15 minutes at 50°C in thermal cycler
    • “Cas9-YcdO”
      • 1.34 µL backbone
      • 0.14 µL insert (Ycdo)
      • 3.52 µL water
      • 5 µL Gibson mix
    • Negative Control
      • 1.34 µL backbone
      • 3.66 µL water
      • 5 µL Gibson mix
    • Positive Control
      • 5µL (+) control
      • 5µL master mix
  • Transformation
    • 1 µL (+) transformation control (CamR)
    • 2 µL Gibson (+) control (AmpR)
    • 2 µL Gibson (-) Control (CamR)
    • 3 µL Gibson Product (CamR)
  • Worked on presentation for 9.2.16
  • Attempted to take inventory of Gibson products (check -20°C inventory spreadsheet)