Team:Paris Bettencourt/Notebook

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Notebook
June 2013
Mo Tu We Th Fr Sa Su
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
July 2013
Mo Tu We Th Fr Sa Su
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
August 2013
Mo Tu We Th Fr Sa Su
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
September 2013
Mo Tu We Th Fr Sa Su
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29

October 2013
Mo Tu We Th Fr Sa Su
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31 1 2 3

Contents

Week 01: 3th - 9th June

Monday 3th June

Tuesday 4th June

Wednesday 5th June

Thursday 6th June

Friday 7th June

Week 02: 10th - 16th June

Monday 10th June

Tuesday 11th June

Wednesday 12th June

Thursday 13th June

Friday 14th June

Week 03: 17th - 23th June

Monday 17th June

Tuesday 18th June

Wednesday 19th June

Thursday 20th June

Friday 21th June

Week 04: 24th - 30th June

Monday 24th June

Trojan Horse

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Starting bacterial culture of MG1655-6300 (Chantal’s glycerol).
Streaking 2 Agar plate with it.

Tuesday 25th June

Trojan Horse

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Choosing a nice colony and launching overnight culture 37° in LB.

Wednesday 26th June

Thursday 27th June

Trojan Horse

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Making MG1655-6300 competent (edit 28/06: failure).

Friday 28th June

Trojan Horse

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sRNA design

PICTURE OF THE DESIGN

Red sequences:

pACYCDuet-1 (CmR) : ATATCCAGTGATTTTTTTCTCCAT
pCOLADuet-1 (KanR) : CGTTTCCCGTTGAATATGGCTCAT

We decided that targeting only two different antibiotic resistance will be enough for a proof of concept. We give up AmpR because otherwise they might be experimental issues with AmpR that is also on phagemid litmus28i.

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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Trojan Horse

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TB-ception

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Human Practices

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Modeling

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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

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Trojan Horse

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 using Electroporation protocol
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)

TB-ception

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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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Transformation results
Negative controls are negative.
Transformed cells grew on plates.
In the evening launch of 5mL Lb cultures from clones on the plates.

Igem Buffer for chemical competent cells.
Igem protocol for chemical competent cells.

Launch overnight culture of NEB turbo.
5mL LB inoculated with a clone from plate of drug team.

TB-ception

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Human Practices

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Modeling

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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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Glycerols of the overnight cultures of the transformation.

  • sT002 : MG1655 pCOLADuet-1
  • sT003 : MG1655 pACYCDuet-1

Making stocks of electro competent NEB turbo
Prepare 10% glycerol solution
Put on Ice 1L of Sterile water, 10% glycerol solution and 10 falcon tubes
Cool down centrifuge to 4°C
Prepare a rack with 50 microtubes at -80°C

Making electroconpetent cells stock
Dilute in 500 mL LB (in a 1000mL flask) 500 ul of NEB turbo pre cultured
Check the OD until it reached 0,5
Put 500mL cultures in 10 50mL falcon tubes in ice
Wait for 20 minutes for the culture to cool down
Centrifuge at 3400 rpm 4°C for 10 minutes
Take out liquid and resuspend in 50 mL ice cold water (resuspend content of two tubes in one tube)
Centrifuge 10 minutes, 4°C , 3400 rpm
Take out liquid and resuspend in 25 mL ice cold water
Centrifuge 10 minutes, 4°C, 3400 rpm
Resuspend all the content of the tubes in 20mL ice cold glycerol
Centrifuge 10 minutes, 4°C, 3400rpm
Resuspend in 5mL ice cold 10°C glycerol solution
Aliquot in 50 tubes (100uL per microtubes)
Store at -80°C

TB-ception

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Human Practices

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Modeling

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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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Human Practices

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Modeling

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Week 06: 8th - 14th July

Monday 8th July

Drug Screening

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Phage Sensor

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Trojan Horse

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Attempting to the SB6.

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Human Practices

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Modeling

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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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Attempting to the SB6.

TB-ception

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Human Practices

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Modeling

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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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Attempting to the SB6.

TB-ception

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Human Practices

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Modeling

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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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Human Practices

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Modeling

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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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Human Practices

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Modeling

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Week 07: 15th - 21th July

Monday 15th July

Drug Screening

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Phage Sensor

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Trojan Horse

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Primer Design

Design primers to PCR KanR out of Duet and LacZalpha out of pUC18 to then clone them into the DUET containing Chloramphenicol

Geneious files in the dropbox: LacZ primers, KanR primers

TB-ception

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Human Practices

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Modeling

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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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Human Practices

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Modeling

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Wednesday 17th 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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Human Practices

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Modeling

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Thursday 18th 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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Human Practices

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Modeling

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Friday 19th 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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Modeling

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Week 08: 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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Human Practices

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Modeling

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Tuesday 23th 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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Human Practices

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Modeling

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Wednesday 24th 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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Human Practices

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Modeling

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Thursday 25th 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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Modeling

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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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Modeling

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Week 09: 29th July - 4th August

Week 10: 5th - 11th August

Week 11: 12th - 18th August

Week 12: 19th - 25th August

Week 13: 26th August - 1st September

Week 14: 2nd - 8th September

Week 15: 9th - 15th September

Week 16: 16th - 22th September

Week 17: 23th - 29th September

Week 18: 30th September - 6th October

Week 19: 7th - 13th October

Week 20: 14th - 20th October

Week 21: 21th - 27th October

Week 22: 28th October - 3th November

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
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team2013@igem-paris.org
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