Haynes Lab:Notebook/ASU iGEM

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==Project Description/Abstract==
==Project Description/Abstract==
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[[#THAT| '''Team Chimeric Reporter:''']]<br>
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'''Team Chimeric Reporter'''<br>
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* Hyder Hussain
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* Hyder Hussain: Topo target plasmid design and building
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* Abhinav Markus
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* Abhinav Markus: (new) Split beta gal assay development for the chimeric reporter (biotinylation proof of concept model)
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* Ryan Muller
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* Maddie Sands: (new) Split beta gal assay development for the chimeric reporter (biotinylation proof of concept model)
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* Ethan Ward
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* Ryan Muller: Inducible Topo chimeric protein production
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[[#THIS| '''Team Cell Surface Reporter:''']]<br>
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* Ethan Ward: Inducible Topo chimeric protein production
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* Nisarg Patel
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* Ryan Muller
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* Rohit Rajan
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'''Team Cell Surface Reporter'''<br>
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* Ellen Qin
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* Nisarg Patel: Split beta gal assay development for the magainin reporter
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* Melinda Jenner
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* Ellen Qin: Magainin fusion protein BioBricking
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* Joe Barth
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'''Support'''<br>
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* Rohit Rajan: Buffers, media (and quality control), preparation of BioBrick registry submissions
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* Melinda Jenner: Buffers, media (and quality control), preparation of BioBrick registry submissions
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* Amanda Ispas: Buffers, media (and quality control), preparation of BioBrick registry submissions
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==BioBricks==
==BioBricks==
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*14. His-Tag + 10aa GS Linker + Bgal fragment (1-w)
*14. His-Tag + 10aa GS Linker + Bgal fragment (1-w)
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==Notes==
 
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===6-7-12===
 
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* Transformation (LSE)
 
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:* Transformed DNA:
 
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::* lacZ (well 4:12G, I732019)
 
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::* p + lacO (well 1:6G, R0011)
 
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:* Cells: neb10beta (donated)
 
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:* Protocol from: http://www.neb.com/nebecomm/products/productc3019.asp
 
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:* Controls: puc19, no DNA (8 plates)
 
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===6-8-12===
 
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* Transformation results:
 
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:* puc19: growth
 
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:* negative control: no growth
 
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:* lacZ, lacO: possible small colonies
 
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* liquid culture in amp media (100 ug / ml):
 
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:* no growth of lacZ, lacO
 
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:* growth of puc19
 
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===6-12-12===
 
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*DH5a Competent Cell Prep
 
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:*Streak plated cells on LB no amp plate, let grow overnight
 
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===6-13-12===
 
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* Transformation (LSE)
 
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:* Transformed DNA:
 
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::* lacZ (well 4:12G, I732019)
 
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::* p + lacO (well 1:6G, R0011)
 
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:* Cells: DH5 alpha (donated)
 
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:* Protocol from: http://openwetware.org/wiki/Haynes:Assembly101 (30 minute transformation)
 
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:* Controls: puc19, no DNA (8 plates)
 
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* Transformation (istb4, Abhi)
 
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:* Transformed DNA:
 
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:* Cells:
 
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:* Protocol from:
 
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:* Controls:
 
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*DH5a Chemically Competent cell prep
 
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:*Grew 2 seed colonies from streak plate in LB no amp
 
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:*Grew controls to test for contamination
 
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::*Both Seed colonies grew, no contamination present
 
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===6-14-12===
 
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*Competent cell prep
 
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:*Prepared CaCl<sub>2</sub> buffer solution and CaCl<sub>2</sub> glycerol buffer solution
 
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:*Grew seed colony in 400mL LB no amp
 
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===6-15-12===
 
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*Competent cell prep
 
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:*Centrifuged falcon test tubes containing liquid colonies
 
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:*Resuspended in CaCl<sub>2</sub> buffer solution and incubated for 15 mins
 
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:*Centrifuged and resuspended in CaCl<sub>2</sub> glycerol buffer solution
 
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:*Chilled overnight
 
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===6-16-12===
 
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*Competent cell prep
 
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:*Aliquotted 200uL into test tubes
 
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:*Stored in -80C
 
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===6-17-12===
 
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:*Streak plated prepared competent cells on LB no amp plate
 
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::*Colonies observed
 
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===6-19-12===
 
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*Transformation (LSE)
 
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:*Transformed DNA:
 
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::*T7 promoter BBa_I712074
 
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::*Constitutive promoter BBa_J23102
 
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:* Cells: DH5 alpha (donated)
 
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:* Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
 
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:* Controls: puc19, no DNA
 
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:* Plated 2 copies of each (100 ul, 250 ul) on LB amp plates.
 
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* Made 50 LB Amp plates.
 
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===6-20-12===
 
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* Plated negative control on LB Amp plate
 
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* Liquid cultures of T7 promoter and constitutive promoter
 
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*Transformation (LSE)
 
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:*Transformed DNA:
 
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::*RBS (well 1:1H BBa_B0030)
 
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::*TetR GFP (well 2:8A Part:BBa_I13522)
 
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:* Cells: DH5 alpha (donated)
 
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:* Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
 
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:* Controls: puc19, no DNA
 
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===6-21-12===
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==Science Resources==
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*Made Liquid Cultures of E.coli transformed with RBS B0030
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* [http://biologylabs.utah.edu/jorgensen/wayned/ape/ A Plasmid Editor (ApE)]: freeware for viewing, editing, annotating, translating, and finding restriction sites in DNA sequences
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*Made Liquid Cultures of E.coli transformed with TetR GFP
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* [http://gcat.davidson.edu/igem10/ Oligator]: tool for creating BioBricks using oligo assembly
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*miniprepped and nanodropped T7 promoter BBa_I712074 and Constitutive promoter BBa_J23102 liquid cultures
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* [http://www.rcsb.org/pdb/home/home.do Protein Data Bank (PDB)]: search for, view, and download full atomic coordinate data for 3D structures of proteins and other molecules. Get tips on editing Jmol 3D images at the [http://openwetware.org/wiki/Haynes:Jmol_Guide Haynes Lab OWW site].
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* liquid cultures:
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* [http://openwetware.org/wiki/Haynes:Protocols Protocols - Haynes Lab]: some protocols from the Haynes Lab
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:* RBS1
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:* RBS2 (duplicate_
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:* GFP1
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:* puc19
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:* negative controls
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:* 5 ml lb amp
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:* overnight cultures
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* replated GFP1 & 2 (duplicates)
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*Nanodropped plasmid DNA samples
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:*Constitutive promoter 1: __ng/uL
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:*Constitutive promoter 2: __ng/uL
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:*T7 promoter 1: __ng/uL
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:*T7 promoter 2: __ng/uL
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===6-22-12===
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==General Lab Links==
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:*Miniprepped liquid cultures: RBS and GFP
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* [https://docs.google.com/spreadsheet/ccc?key=0AghpgNgJLx-ldGtGSUhRRjFRQ0VCdlo4WXdyNlVwWVE Ordering Spreadsheet]
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===6-25-12===
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==Safety==
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===6-26-12===
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* [https://cfo.asu.edu/ehs Environmental Health & Safety at ASU]
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===6-27-12===
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* [http://ehsaweb.asu.edu/ Individual Training Record]: To check your ASU individual safety training record, log in at the EHS Assistant page. Click the "Individual Training Record" link and then enter your ASU ID.
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===6-28-12===
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__NOTOC__
__NOTOC__

Current revision

ASU iGEM 2012

Search this Project

Customize your entry pages

Project Description/Abstract

Team Chimeric Reporter

  • Hyder Hussain: Topo target plasmid design and building
  • Abhinav Markus: (new) Split beta gal assay development for the chimeric reporter (biotinylation proof of concept model)
  • Maddie Sands: (new) Split beta gal assay development for the chimeric reporter (biotinylation proof of concept model)
  • Ryan Muller: Inducible Topo chimeric protein production
  • Ethan Ward: Inducible Topo chimeric protein production


Team Cell Surface Reporter

  • Nisarg Patel: Split beta gal assay development for the magainin reporter
  • Ellen Qin: Magainin fusion protein BioBricking


Support

  • Rohit Rajan: Buffers, media (and quality control), preparation of BioBrick registry submissions
  • Melinda Jenner: Buffers, media (and quality control), preparation of BioBrick registry submissions
  • Amanda Ispas: Buffers, media (and quality control), preparation of BioBrick registry submissions


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)


Science Resources

General Lab Links

Safety
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