Haynes Lab:Notebook/ASU iGEM

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Project Description/Abstract

Team Chimeric Reporter:

  • Hyder Hussain
  • Abhinav Markus
  • Ryan Muller
  • Ethan Ward

Team Cell Surface Reporter:

  • Nisarg Patel
  • Ryan Muller
  • Rohit Rajan
  • Ellen Qin
  • Melinda Jenner
  • Joe Barth

BioBricks

Magainin Reporter

  • Magainin
  • Magainin + 2xGS Linker + His-Tag
  • Bgal fragment (a-1)
  • Bgal fragment (a-4)
  • Bgal fragment (w)
  • Bgal fragment (1-w)
  • Magainin + 2aa GS Linker + His-Tag + 10aa GS Linker + Bgal fragment (a-1)
  • Magainin + 2aa GS Linker + His-Tag + 10aa GS Linker + Bgal fragment (a-4)
  • Magainin + 2aa GS Linker + His-Tag + 10aa GS Linker + Bgal fragment (w)
  • Magainin + 2aa GS Linker + His-Tag + 10aa GS Linker + Bgal fragment (1-w)
  • His-Tag + 10aa GS Linker + Bgal fragment (a-1)
  • His-Tag + 10aa GS Linker + Bgal fragment (a-4)
  • 13. His-Tag + 10aa GS Linker + Bgal fragment (w)
  • 14. His-Tag + 10aa GS Linker + Bgal fragment (1-w)

Notes

6-12-12

  • DH5a Competent Cell Prep
  • Streak plated cells on LB no amp plate, let grow overnight

6-13-12

  • Transformation (LSE)
  • Transformed DNA:
  • lacZ (well 4:12G, I732019)
  • p + lacO (well 1:6G, R0011)
  • Transformation (istb4, Abhi)
  • Transformed DNA:
  • Cells:
  • Protocol from:
  • Controls:
  • DH5a Chemically Competent cell prep
  • Grew 2 seed colonies from streak plate in LB no amp
  • Grew controls to test for contamination
  • Both Seed colonies grew, no contamination present

6-14-12

  • Competent cell prep
  • Prepared CaCl2 buffer solution and CaCl2 glycerol buffer solution
  • Grew seed colony in 400mL LB no amp

6-15-12

  • Competent cell prep
  • Centrifuged falcon test tubes containing liquid colonies
  • Resuspended in CaCl2 buffer solution and incubated for 15 mins
  • Centrifuged and resuspended in CaCl2 glycerol buffer solution
  • Chilled overnight

6-16-12

  • Competent cell prep
  • Aliquotted 200uL into test tubes
  • Stored in -80C

6-17-12

  • Streak plated prepared competent cells on LB no amp plate
  • Colonies observed

6-19-12

  • Transformation (LSE)
  • Transformed DNA:
  • T7 promoter BBa_I712074
  • Constitutive promoter BBa_J23102
  • Made 50 LB Amp plates.

6-20-12

  • Plated negative control on LB Amp plate
  • Liquid cultures of T7 promoter and constitutive promoter
  • Transformation (LSE)
  • Transformed DNA:
  • RBS (well 1:1H BBa_B0030)
  • TetR GFP (well 2:8A Part:BBa_I13522)

6-21-12

  • Made Liquid Cultures of E.coli transformed with RBS B0030
  • Made Liquid Cultures of E.coli transformed with TetR GFP
  • miniprepped and nanodropped T7 promoter BBa_I712074 and Constitutive promoter BBa_J23102 liquid cultures
  • liquid cultures:
  • RBS1
  • RBS2 (duplicate_
  • GFP1
  • puc19
  • negative controls
  • 5 ml LB amp
  • overnight cultures
  • replated GFP1 & 2 (duplicates)
  • Nanodropped plasmid DNA samples
  • Constitutive promoter 1: __ng/uL
  • Constitutive promoter 2: __ng/uL
  • T7 promoter 1: __ng/uL
  • T7 promoter 2: __ng/uL

6-22-12

  • Miniprepped liquid cultures: RBS (well 1:1H BBa_B0030) and TetR GFP (well 2:8A Part:BBa_I13522)

6-26-12

  • Transformation:
  • Transformed DNA:
  • double terminator (B0017, 2:6K)
  • T7 RNA polymerase (I715038, 2:15C)
  • puc19, negative control

6-27-12

  • 6-26 transformation results:
  • Controls correct
  • 2x terminator: ~19 colonies
  • RNA pol: 1 colony
  • Liquid cultures including controls

6-28-12

  • Miniprepped double terminator (B0017, 2:6K) and T7 RNA polymerase (I715038, 2:15C) liquid cultures

7-2-12

  • Cleaned up liquid waste
  • Made SOB media
  • Finalized oligos for magainin construct

7-3-12

  • Autoclaved SOB media
  • Added glucose to make SOC media
  • Nanodropped double terminator (B0017, 2:6K) [DT1: 24.5, DT2: 29.6] and T7 RNA polymerase (I715038, 2:15C) [P1: 64.6, P2: 55.3] liquid cultures


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