Team:UNIPV-Pavia/Notebook/Week5Sep

From 2009.igem.org

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B9new-1 - B9new-2 - B9new-3 - B9new-4 - B9new-5 - A19-1 - A19-2 -  - Marker-  - A11 -  - GFP3 -  -  -  
B9new-1 - B9new-2 - B9new-3 - B9new-4 - B9new-5 - A19-1 - A19-2 -  - Marker-  - A11 -  - GFP3 -  -  -  
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[[Image:GEL021009.jpg|thumb|500px|left|Screening gel for B9new 1-5, A19-1 and A19-2. Gel extraction for A11-3 and E0240.]]
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Revision as of 14:16, 2 October 2009

EthanolPVanimation.gif

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 September 28th, to October 4th, 2009

Previous Week Next Week

September, 28th

Top

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.


Top

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.


Top

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.

Top

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 - - -


File:GEL021009.jpg
Screening gel for B9new 1-5, A19-1 and A19-2. Gel extraction for A11-3 and E0240.

Top

October, 3rd

Top

October, 4th

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