Wisconsin-Madison/30 June 2009

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