Team:Paris Bettencourt/Notebook

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Notebook
June 2013
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10 11 12 13 14 15 16
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24 25 26 27 28 29 30
July 2013
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14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
August 2013
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September 2013
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9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29

October 2013
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21 22 23 24 25 26 27
28 29 30 31 1 2 3

Contents

Week 05: 1st - 7th July

Monday 01st July

Drug Screening

Media was made, Strains sD001-sD004 were received, inoculated, and put into stock and catalog.

4 strains were received from Jake:

  • E. coli: BL21 (DE3) ko20 ΔcysI, Δfpr, ΔydbK
  • E. coli: NEBTurbo zmSIR Chloramphenicol
  • E. coli: NEBTurbo zmFNR Spectinomycin
  • E. coli: NEBTurbo soFD, zmSIR Chloramphenicol
Media and Glycerol stock were prepared:

Media Preparation
3 500ml bottles of LB broth and LB agar were prepared by standard methods.
12.5g/500ml powder/water for broth and 20g/500ml powder/water for agar.
Bottles were autoclaved. Additionally 1 flask of 500ml of LB broth was made in the same fashion.

Glycerol Stocks
Single colonies from agar plates were picked and used to innoculate 5ml LB broth overnight.
750ml of overnight culture was added to 250ml of 60% glycerol in a cryotube.
Two sets of Glycerol stocks were used for each of sD001, sD002, sD003, sD004; one set was frozen at -20ºC and the other set was frozen at -80ºC.

Phage Sensor

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Tuesday 02st July

Drug Screening

Plasmids were extracted from strains sD002, sD003, sD004

Plasmid Extraction
Plasmids pD004 (pCDF.ew12 zmFNR Spectinomycin), pD005 (pACYC.ew13 soFD, zmSIR Chloramphenicol), pD006 (pACYC.ew17 zmSIR Chloramphenicol) were extracted from NEBTurbo cells using a Thermo Scientific GeneJet Plasmid mini prep kit as described in the protocol (available on google drive).
Lacking Resuspention solution we used some from a different mini prep upstairs.
Plasmids were eluted in 100ul of nanopure water and frozen at -20ºC. Plasmids were included in catalog.

Phage Sensor

Glycerol stock was made and cells were transformed.

glycerol stock
From overnight culture of MG1655-6300 O/N : T001
Centrifuge 4000rpm, 10 minutes,
Take out liquid
resuspend cells in 1mL glycerol, 2mL LB
separate in two cryotubes, one for the -80°C, one for the -20°C

Electroporation
Making MG1655-6300 competent (Aude?s protocol for electrocompetent cells & electroporation [ref needed])
test of the competent cells (negative
transforming with pCOLADuet-1
transforming with pACYCDuet-1
plating 3 different quantity of cells (20ul, 50uL, 100uL) respectively on Cm and Kan plates (dilution 1000x for the antibiotics)

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Wednesday 03st July

Drug Screening

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Competent Cells : BL21 (DE3) dCysI dFpr dydbk

  • We grew 5ml of sD001 cells overnight from a single colony.
  • This 5ml was used to innoculate 500ml of LB broth.
  • Broth was incubated for 1.5h and optical density was read at 0.62 OD600.
  • Cells were alloquoted into 10 falcon tubes (50ml each) and centrifuged at 4000xg for 20 minutes at 4C.
  • Supernatant was removed and cells were resuspended in 200ml of Buffer 1 (4x 50ml) and left on ice for 20 minutes.
  • Cells were centrifuged again for 20 minutes at 4000xg.
  • Supernatant was removed and cells were resuspended in 12ml Buffer 2.
  • Cells were alloquoted at 500ul into microcentifuge tubes and frozen at -80C.
Buffer 1: 50mM CaCl2
Buffer 2: 0.53ml 2M CaCl2 2.8ml 60% Glycerol 8.67ml sterile H2O

Phage Sensor

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Trojan Horse

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TB-ception

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Thursday 04st July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Friday 04st July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Week 03: 8th - 14th July

Monday 8th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Tuesday 9th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Wednesday 10th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Thursday 11th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Friday 12th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Week 03: 15th - 21th July

Monday 15th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Tuesday 16th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Wednesday 17th July

Drug Screening

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Phage Sensor

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Thursday 18th July

Drug Screening

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Phage Sensor

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Friday 19th July

Drug Screening

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Phage Sensor

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Week 04: 22th - 28th July

Monday 22th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Tuesday 23th July

Drug Screening

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Phage Sensor

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TB-ception

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Wednesday 24th July

Drug Screening

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Phage Sensor

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Thursday 25th July

Drug Screening

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Phage Sensor

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Friday 26th July

Drug Screening

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Phage Sensor

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Trojan Horse

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TB-ception

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Week 05: 29th July - 4th August

Week 06: 5th - 11th August

Week 07: 12th - 18th August

Week 08: 19th - 25th August

Week 09: 26th August - 1st September

Week 10: 2nd - 8th September

Week 11: 9th - 15th September

Week 12: 16th - 22th September

Week 13: 23th - 29th September

Week 14: 30th September - 6th October

Week 15: 7th - 13th October

Week 16: 14th - 20th October

Week 17: 21th - 27th October

Week 18: 28th October - 3th November

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
team2013@igem-paris.org
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