Team:UNIPV-Pavia/Notebook/Week2Oct

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December 2008
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March 2009
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April 2009
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May 2009
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June 2009
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July 2009
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August 2009
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September 2009
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October 2009
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Week from October 5th, to October 11st, 2009

Previous Week Next Week

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.

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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.

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October, 7th

pH sensor

Qui risultati?


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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.


Top

October, 9th

  • Sequencing results for:
    • A19-1 sequence ok!
    • A19-2 very noisy (NNNNN)
  • 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)
    • B5new2-3
    • F2620TOP10
    • A21
    • A21 + 3OC6HSL 1uM
  • in 8 ml of LB + indicated antibiotic + 2% glucose and incubated them at 37°C, 220 rpm for 24 hours.
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.


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October, 10th

pH sensor

Qui risultati?


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October, 11th

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