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 70 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 falcon 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 doens'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.

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

• Prepartion of 2 falcons 50ml containing 35ml LB+Amp+2%glucose and infected from glycerol stocks with B9 and F2620. They have been incubated overnight at 37°C statically, without shaking.

pH sensor

• Qui risultati?
• Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:
  • RBS33
  • A2
  • pNhaA + GFP (from now on it we'll be indicated only as pNhaA)
• Overnight incubation of the three cultures.

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: A9-B5
• B9: F2620-B5
• B12: J118-B5
• B13: J106-B5
• A19: F2620-4Cm5

Inoculus of pNhaA and RBS32 for pH test, tomorrow will they will be diluted in 4 different LB, with different pH inoculus 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 3OC6HSLnM. Tomorrow we will see if the part works.

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

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

October, 1st

• From the cultures incubated yesterday, F2620 grew, but B9 didn't. We have decided not to induce anymore, because F2620 have an important leakage.

Yesterday ligations have been inoculate in 1ml Lb + AB from colony and incubated overnight at 37° 220rpm. The coltures 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 inoculus 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 incolus from glycerol stocks of

• A11
• GFP3

to repeat ligation tomorrow.

Yesterday inoculus of pNhaA and RBS32 have been diluted 1:100 in 4 new cultures, using medium with pH:

• 5,5
• 6,6
• 7,5
• 8,5

• 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. Inoculus of A8, A2, RBS32 for tomorrow induction test of constitutive (?) promoter A8 with 3OC6HSL. Inoculus of A15 for tomorrow lysis verific.

October, 2nd

At TECAN: pH test on cultures grown in LB at pH:

• 5,5
• 6,6
• 7,5
• 8,5

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

October, 4th

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