From 2009.igem.org
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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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November 2009
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Week from October 5th, to October 11st, 2009
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.
pH sensor
- Infection of 4 ml LB + Amp with 8 ul from glycerol stock of:
- 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 21 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:
- 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.
- 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.
Experiment with Tecan F200