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Wisconsin-Madison/30 June 2009
From 2009.igem.org
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June 30, 2009
Ex 17: Salt Induced ProU GFP tests
Test 4 Results: Plate Reader
- BAD RESULTS
- OD’s look OK except for water and LB
- Everything Fluoresces….
Messed UP – contamination?
- Could the plate shaking too hard?
- Can bacteria crawl under the plates?
- How full should we fill the wells?
Inoculate: DH10B, ProU only, GFP only, ProU+GFP
Ex 15: Inducing Various Modified (triple) E.Coli Strains
Add layer of Ethyl Acetate – 0.5 mM
(Add this layer when done inducing – after 60 hours)
Questions:
- What kills the cells? Toxicity of Isopentenol and Ethyl Acetate?
- Plate liquid culture of isopentenal before we add Ethyl Acetate. Syd said cells are supposed to die during induction
- Can we test for presence and amount DMAPP and FPP? How?
- Ask Brian for Tet Resistance Empty Vector. Better control for Isopentenol Tests
Ex 16:
MiniPrep of BB (P1) for verification
Digestion with Ecor1
(miniprep of backbone cut by Spe1 and ECOR1 then re-ligated)
Backbone: 2160bp
Red Gene: 860bp
Ex 18: pTaq Induction and experiments
Protein Gel:
PCR Digestion with 2-log ladder:
NOT even template DNA was visable on gel – control fail
- not good expression or bad extraction
- followed exact protocol from paper with same reagents
Brian has sequencing primers that we can use to verify
Will submit pTaq as Biobrick – VERY VERY Valuable BioBrick
MiniPrep: of pTaq
Ex 17: Salt Tests
Inoculated ProU, ProU+GFP, and GFP on pSB1A3 and DH10B culture
Ex 10: Cyanobacteria
