Haynes Lab:Notebook/ASU iGEM

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

6-7-12

• Transformation (LSE)

• Transformed DNA:

• lacZ (well 4:12G, I732019)
• p + lacO (well 1:6G, R0011)

• Cells: neb10beta (donated)
• Protocol from: http://www.neb.com/nebecomm/products/productc3019.asp
• Controls: puc19, no DNA (8 plates)

6-8-12

• Transformation results:

• puc19: growth
• negative control: no growth
• lacZ, lacO: possible small colonies

• liquid culture in amp media (100 ug / ml):

• no growth of lacZ, lacO
• growth of puc19

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)

• Cells: DH5 alpha (donated)
• Protocol from: http://openwetware.org/wiki/Haynes:Assembly101 (30 minute transformation)
• Controls: puc19, no DNA (8 plates)

• 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

• Cells: DH5 alpha (donated)
• Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
• Controls: puc19, no DNA
• Plated 2 copies of each (100 ul, 250 ul) on LB amp plates.

• 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)

• Cells: DH5 alpha (donated)
• Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
• Controls: puc19, no DNA

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

• Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
• Cells: dh5a

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
• 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