Week from September 28th, to October 4th, 2009

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September, 28th

pH sensor

3rd experiment

• We put 50 ul into 5 ml LB NaCl 171 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four hours and a half.
• OD measure of the three falcons with TECAN in order to make dilutions with a final volume of 3 ml and an OD equal to 0,02.
• We diluted:
  • RBS into:
    • LB NaCl 171 mM + Amp pH 5,5
    • LB NaCl 171 mM + Amp pH 6,6
    • LB NaCl 171 mM + Amp pH 7,5
    • LB NaCl 171 mM + Amp pH 8,5
  • pNhaA into:
    • LB NaCl 171 mM + Amp pH 5,5
    • LB NaCl 171 mM + Amp pH 6,6
    • LB NaCl 171 mM + Amp pH 7,5
    • LB NaCl 171 mM + Amp pH 8,5
  • A2 into:
    • LB NaCl 171 mM + Amp pH 5,5
    • LB NaCl 171 mM + Amp pH 6,6
    • LB NaCl 171 mM + Amp pH 7,5
    • LB NaCl 171 mM + Amp pH 8,5
• We started this 6 hours' experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.

September, 29th

• A14 induction test using IPTG

The part doesn't show any induction!! :-( To test what happened, we have inoculated A14 L-1 from glycerol stocks. tomorrow it will be diluted 1:100 in 2 falcons (5ml), one of wich will be induced 10nM.

• Inoculum of T9002 from glycerol stocks in 2 falcon tubes, one grown anaerobically and the other areobically. They have been induced with 3OC6HSL 10nM and next morning the measure of GFP was about the half in the anaerobically grown culture respect to the aerobically grown one.

• Preparation of 2 falcon tubes (50 ml) containing 35ml LB+Amp+2%glucose and infected from glycerol stocks with B9-2 and F2620MIT2. They have been incubated overnight at 37°C without shaking.

Experiment with Tecan F200

• Download Protocol

September, 30th

We watched B9 and F2620 grown overnight in anaerobic way. In B9 lots of foam has developed that we couldn't see in F2620. Is it for it fermented and made CO2? In the evening they have been pelletted and F2620's pellet was bigger than B9's one.

LIGATIONS:

• B7 = A4(E-S) + B5new2-3(E-X) in pSB1AK3
• B9: F2620(E-S) + B5new2-3(X-P-ClaI) in pSB1A2
• B12: J23118(S-P) + B5new2-3(E-X) in pSB1A2
• B13: J23106(S-P) + B5new2-3(E-X) in pSB1A2
• A19: F2620(E-P) + 4C5(E-P) in pSB4C5

Inoculum of A15-1, A15-3 F2620 from glycerol stocks for tomorrow lysis test. A15-3 has also been plated on LB+amp+agar2% to pick single colony for tomorrow test.

A14 inoculated yesterday has been diluted 1:100 into 2 falcons and induced with 1 mM IPTG. Tomorrow we will see if the part works.

• Inoculum of 15ul from glycerol stock of B9 and F2620 in 2 cultures each, one closed (anaerobic) and the other a little opened ("aerobic"). The cultures have been induced immediately with 3OC6HSL 10nM.

They have been incubated overnight at 37°C without shaking.

Experiment with Tecan F200

• Download Protocol

October, 1st

• From the cultures incubated yesterday, F2620 grew, but B9 didn't. We have decided not to induce anymore and to exploit F2620 leakage to express pdc and adhB.

Yesterday ligations have been inoculated in 1ml LB + AB from colonies and incubated at 37° 220rpm for about 6 hours. Then glycerol stocks were prepared and the remaining 250 ul of the cultures have been re-filled with 5 ml of LB + antibiotic and incubated overnight to prepare the screening. The cultures are:

• B7-1 (Amp)
• B7-2 (Amp)
• B12-1 (Amp)
• B12-2 (Amp)
• B13-1 (Amp)
• B13-2 (Amp)
• B9new-1 (Amp)
• B9new-2 (Amp)
• B9new-3 (Amp)
• B9new-4 (Amp)
• B9new-5 (Amp)
• A19-1 (Cm)
• A19-2 (Cm)

Dilution 1:100 of cultures:

• A15-1
• A15-3
• F2620

and inoculum of A15-3 colony picked from colony grown on plate infected yesterday. Cultures have been incubated for further 4 hours for lysis test at TECAN in the afternoon.

• Preparation of 3OC6HSL at desired concentration, to be used for induction test in the afternoon.

A14 L-1 induced and A14 L-1 NOT induced have been measured using TECAN: the fluorescence in green wasn't significantly different. It could mean that A14 has mutated, so we have prepared an inculum from glycerol stocks of

• A11
• E0240

to repeat ligation tomorrow.

• Preparation of LB+Amp 10% glucose:

LB + Amp has been prepared as always for 500ml, but just 400ml of water have been added. The further 100ml have been used to dilute 50g glucose and have been added after autoclave, filtering them.

• Preparation of LB+Amp (500ml)

TECAN test in the afternoon for A15 lysis. Glycerol stock for all the cultures incubated this morning. Inoculum of A8pg, A2, RBS32 for tomorrow induction test of A8pg construct with aTc. Inoculum of A15-3 for tomorrow lysis test (to repeat today's test because the results were not satisfying).

Experiment with Tecan F200

• Download Protocol
• Download Protocol

October, 2nd

Dilution of A15 and induction with 3OC6HSL to test the activity os this part (it didn't work yesterday!)

In the morning we have miniprepped:

• A11-3
• GFP3

to re-ligate A14

We have also miniprepped:

• B9 new-1
• B9 new-2
• B9 new-3
• B9 new-4
• B9 new-5
• A19-1
• A19-2

incubated overnight at 37°C 220rpm. They have been refilled from yesterday cultures used for glycerol stocks.

After mini-prep, the cultures have been digested:

• A11-3 (S-P) for ligation
• GFP3 (X-P) for ligation
• B9 new-1 (E-P) for screening
• B9 new-2 (E-P) for screening
• B9 new-3 (E-P) for screening
• B9 new-4 (E-P) for screening
• B9 new-5 (E-P) for screening
• A19-1 (E-P) for screening
• A19-2 (E-P) for screening

Gel has been filled as follows: B9new-1 - B9new-2 - B9new-3 - B9new-4 - B9new-5 - A19-1 - A19-2 - - Marker- - A11 - - GFP3 - - -

October, 3rd

• Cloning
• Wiki updating

October, 4th

• Wiki updating

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