Wisconsin-Madison/28 June 2009

June 28, 2009
'''Ex 17: GFP Regulation by ProU Promoter

Test 3: Results: Fail, problems with the Plate Reader (connected to simulator)



'''Ex 15: Inducing Various Modified (triple) E.Coli Strains

Induction: 	1. Irrabinose -  0.8 – 1% g/100mL

2. Wait 30 minutes

3. 1mM conc. IPTG (100uL/100mL)

4. Layer of Dodecane to trap isopentenol

Placed 5 ml of sample to into fresh 100ml LB, 50uL Amp, 73.5uL Cm, 100uL Tet OD 600

'''Control / BL21 (DE3) / MG1655 (W) / MG1655 Delta Ara

.395 / .290 / .184 / .249

.364 / .246 / .174 / .211

.398 / .266 / .183 / .227

.425 (I) / .225 / .219 / .227

.564 / .240 / .214 / .229

.916 / .305 / .239 / .303

1.35 / .322 / .33 / .332

2.039 / .429 (I) / .427 (I) / .432 (I)

'''1.0087 / 1.048 / 1.086 / 1.0085

'''40uL IPTG solution? (0.002g/ culture)

Results: It took 2 hours to reach OD

Maybe IPTG and Arabinose kill cell

Maybe Arabinose slows maetablolis,

Note - Cultures grew extraordinarily fast – USE less see culture (2.5ml)

Ex 16: MiniPrep of Choline Colonies A,C

Ran on 1% Agarose Gel

Undigested (A, C), 1kb Ladder, Digested (A, C)

Pc: PEAMT cut

Pu: PEAMT uncut

Expected size: 4150bp

Cut with: Pst1

'''Ex 16:

Growth:

Streaked and Inoculated: multiple colonies from multiple plats for Miniprep tomorrow

Both iGEM Plasmid: pSB1A3 different digestions

P1 - cut Ecor1and Spe1

P2 - cut Xba1 and Pst1

Also transformed NudF an ProU from last ligation :



Ex; 	Absorbance and fluorescence taken, man

'''Autoclaving Medium:

-	Standard is to fill bottle half way full

-	It is OK to fill bottle full unless Agar

-	Agar needs to be half, will bubble

-	Better to make half bottle to avoid contamination of medium

'''Ex 10: Cyanobacteria

added 8mL to 2mL 6803 cultures to achieve 10mL cultures - NP