Tuesday 28th July

Lab Work

Plasmid extraction

by Coralie

Biobricks:

• BBa_K1707005 #1 and #2
• BBa_K1707006 #1 and #2
• BBa_K1707007 #1 and #2

With Macherey-Nigel Nucleospin kit

Digestion

by Coralie

• BBa_B0030 and BBa_R0051
  • 2µL Buffer FastDigest 10x
  • 1µL SpeI
  • 1µL PstI
  • 10µL Plasmid
  • 6µL H2O

• BBa_K1707007, BBa_K1707006, BBa_K1399019 and BBa_K1399023
  • 2µL Buffer FastDigest 10x
  • 1µL XbaI
  • 1µL PstI
  • 10µL Plasmid
  • 6µL H2O

• BBa_J23117
  • 1µL Buffer FastDigest 10x
  • 1µL XbaI
  • 1µL PstI
  • 2µL Plasmid
  • 5µL H2O

Incubation 1h30, 37°C

Purification gel

by Pauline, Audrey and Coralie

Biobricks:

• BBa_K1707006
• BBa_K1707007
• BBa_K1399019
• BBa_K1399023

Purification on agarose gel 1%, migration 100V Cut with a scalpel Purification with the PCR Clean up kit from Macherey-Nagel Observation: BBa_K1399019 and BBa_K1399023 weren't on the right length on the gel

Soil experiment

by Audrey, Johan and Coralie

Soil

We put 100µL of each culture on MacConkey plate with specific antbiotic (Control +):

• 1696: Tetracyclin
• 1693: Spectinomycine
• 1320: Chloramphenicol

We put 30 mL of each culture in 60g of non sterile soil in a pot.

• 1696: 3 pots
• 1693: 3 pots
• 1320: 3 pots

We put 30 mL of clean LB in 60g of non sterile soil in a pot (Control -)

• LB: 3 pots

After this contamination, we take 1g of each pot to make the J0 measurement. We dilute it in 5mL of sterile H2O, and shake it during 5 min. Then, we let it to decant during 15 min. We take 50µL of the supernatant, and dilute it in 950µL of Sterile H2O (complete dilution from the soil is now at 10-2) We put 100µL of this previous dilution on plates that fits. We incubate plates over night at 37°C

Pots are placed in trays: 1 strain by tray. Trays are covered by a shrink-wrap, and we let them in room temperature

Water

We test two different water: seawater, from the Atlantic ocean, and stagnant water from Orsay's pool. For each water, we make dilutions: 10-1, 10-2, 10-3 and 10-4 We 100µL of different dilution on each type of plate:

• LB without antibiotic
• LB + Spectinomycin
• LB + tetracyclin
• LB + Chloramphenicol
• MacConkey without antibiotic
• MacConkey + Spectinomycin
• MacConkey + Tetracyclin
• MacConkey + Chloramphenicol

We incubate plates over night at 37°C

New culture

by Pauline

Biobricks:

• BBa_K1707008
• BBa_K1707010

We take 2 colony of each, and put it in 5mL of LB + 20µg/mL Chloramphenicol Incubation 37°C, 250 rpm

Purification

by Audrey

Biobricks:

• BBa_B0030
• BBa_R0051
• BBa_J23117 #1 and #2
• BBa_K1399019
• BBa_K1399023
• BBa_K1707006
• BBa_K1707007

With PCR CLean-up/Gel extraction kit from Macherey-Nigel

Quantification

by Audrey

Agarose gel 1% Migration 100V

Interlab study

by Johan

Tecan utilisation :

we use only LB without chloramphenicol and we suspect a contamination of our samples.

We depose in inch well 300µL

We analyse the OD and fluorescence's variation (the excitation and emission wave lenght were choose after a scan in the process to obtain the best results)

For each sample, we depose twelve time (12x8 plate)

• LB
• Competent cells
• Cells with J23101
• Cells with J23101 + GFP
• Cells with J23106
• Cells with J23106 + GFP
• Cells with J23117
• Cells with J23117 + GFP

We let's run for 20 cycles of 1 hour

New culture of BBa_J23101/BBa_J23106/BBa_J23117 + BBa_I13504.

Member present:

• Instructors: Alice
• Students: Coralie, Audrey, Pauline, Johan and Seong-Koo

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