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

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October, 5th

• Miniprep for:
  • A19-2
  • F2620MIT1
  • B5new2-3
  • A8pg

• Digestion for:
  • A19-2(S-P)
  • F2620MIT1(E-S)
  • B5new2-3(X-P-ClaI)
  • A8pg(E-X)

• Gel run/cut/band purification for all.

• Ligations:
  • A20 = F2620(E-S) + A8pg(E-X) in pSB1A2
  • A21 = A19-2(S-P) + B5new2-3(X-P-ClaI) in pSB4C5
• We incubated the ligations at 16°C overnight.

• 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

• We transformed 1 ul of the 1:20 dilutions of A20 and A21 ligations. We incubated the plated bacteria at 37°C overnight.

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

• We inoculated 3 colonies of A20 plate (in 5 ml of LB + Amp) and 4 colonies of A21 plate (in 5 ml of LB + Cm). We incubated these inocula at 37°C, 220 rpm overnight.

October, 8th

• Glycerol stocks, miniprep and 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:
  • F2620TOP10 Amp
  • B3
  • B4
  • 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

• Experiment with Tecan F200 using A1 construct.

• After 24 hours from the inoculum, we inoculated 50 ul of:
  • B9-2
  • B9-2 bis
  • B5new2-3
  • F2620TOP10
  • A21
  • A21 + 3OC6HSL 1uM
• in 30 ml of LB + suitable antibiotic + 10% glucose (anaerobic) in order to begin fermentation. We added 3OC6HSL 1 uM to A21 and 1%w/v of ethanol to B9-2 bis cultures.

• We incubated the fermentations at 37°C, 220 rpm for 24 hours.

• Before incubation, we aliquoted 200 ul of all these cultures (except B9-2 + 1%EtOH) in the microplate reader (samples in triplicate). We incubated them for 24 hours.

• Miniprep for:
  • A19-1
  • B3
  • B4
  • B5new2-3

• Digestion:
  • A19-1 (S-P)
  • B3 (X-P)
  • B4 (X-P)
  • B5new2-3 (X-P-ClaI)

• Gel run: unfortunately B5new2-3 had some undigested bands near the band of interest, so we decided to repeat this digestion on Monday. Moreover, F2620TOP10 had a bright band with high molecular weight (nicked DNA) and the band of interest looked a bit smeared, maybe digestion failed. We decided to repeat this digestion on Monday as well.

• Gel cut for B3(X-P), B4(X-P) and A19-1(S-P). We stored the cut gel at +4°C.

October, 11th

• End of fermentation: we measured the pH and then we centrifuged the falcon tubes at 9000 rpm for 10 minutes. We stored the supernatant at +4°C.

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