Week from October 5th, to October 11st, 2009

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October, 5th

• Team Meeting

pH sensor

• Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:
  • RBS33
  • A2
  • pNhaA
• Overnight incubation of these three cultures at 37°C, 220 rpm.

October, 6th

pH sensor

4th experiment

• We put 50 ul into 5 ml LB NaCl 250 mM + Amp pH 6,6 falcon for each of the overnight cultures RBS33 - A2 - pNhaA and incubated them for four five hours at 37°C, 220 rpm.
• 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 250 mM + Amp pH 5,5
    • LB NaCl 250 mM + Amp pH 6,6
    • LB NaCl 250 mM + Amp pH 7,5
    • LB NaCl 250 mM + Amp pH 8,5
  • pNhaA into:
    • LB NaCl 250 mM + Amp pH 5,5
    • LB NaCl 250 mM + Amp pH 6,6
    • LB NaCl 250 mM + Amp pH 7,5
    • LB NaCl 250 mM + Amp pH 8,5
  • A2 into:
    • LB NaCl 250 mM + Amp pH 5,5
    • LB NaCl 250 mM + Amp pH 6,6
    • LB NaCl 250 mM + Amp pH 7,5
    • LB NaCl 250 mM + Amp pH 8,5
• We started this 24 hours experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.

October, 7th

pH sensor

Qui risultati?

October, 8th

• Digestion screening for A20 (3 samples) and A21 (4 samples): all of them were positive!

• Over-day experiment on beta-gal activity using BioVision Lactose Assay kit: we diluted 1:100 the overnight cultures of B0033, A5 and V1022 in 30 ml of LB + Amp + 4.5% lactose for B0033 and A5 and in 30 ml of LB + 4.5% lactose without antibiotic for V1022. Then we incubated the three cultures (in 50 ml falcon tubes) at 37°C, 220 rpm; every 2 hours we measured the OD600 and the lactose using the commercial kit.

• All the streaked plates were grown and all the unshaken falcon tubes confirmed the phenotypes: B9-2, B9new-1, B9new-2 were cloudy, while B5new2-3 and F2620 were normal. This phenotype was not confirmed on the plates, which looked equal.

pH sensor

• Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:
  • RBS33
  • A2
  • pNhaA
• Overnight incubation of these three cultures at 37°C, 220 rpm.

October, 9th

• Sequencing results for:
  • A19-1 sequence ok!
  • A19-2 very noisy (NNNNN)
• We will try to repeat the A21 assembly using A19-1 instead of A19-2 to be sure that the sequence is correct.

• LB + 2% glucose and LB + 10% glucose preparation (0.5 l for each).

• Experiment on B9-2 and F2620TOP10: we inoculated 8 ul of their glycerol stocks in 8 ml of LB + Amp (without glucose) to see if the strange phenotype of B9 happened in this conditions. We incubated the 15 ml falcon tubes at 37°C without shaking (anaerobic).

• We inoculated 8 ul of these glycerol stocks:
  • B9-2 (X2) Amp
  • B5new2-3 Amp
  • F2620TOP10 Amp
  • A21 Cm
  • A21 + 3OC6HSL 1uM Cm
• in 8 ml of LB + indicated antibiotic + 2% glucose and incubated them at 37°C, 220 rpm for 24 hours.

• We also inoculated 8 ul of these glycerol stocks:
  • A19-1 Cm
  • A1 Amp
• in 5 ml of LB + suitable antibiotic.

pH sensor

5th experiment (with this experiment we try to give E.coli a pH and sodium shock to see if something happens).

• 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 four hours and a half at 37°C, 220 rpm.
• 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 600 mM + Amp pH 10
    • LB NaCl 600 mM + Amp pH 11,2
  • pNhaA into:
    • LB NaCl 600 mM + Amp pH 10
    • LB NaCl 600 mM + Amp pH 11,2
  • A2 into:
    • LB NaCl 600 mM + Amp pH 10
    • LB NaCl 600 mM + Amp pH 11,2
• We started this 6 hours experiment that performed absorbance and fluorescence measures (on three different wells) for each sample every 5 minutes.

October, 10th

• After 24 hours from the inoculum, we inoculated 50 ul of

October, 11th

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