IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/01

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Aleksandra
Glycerols of the yesterday's overnight cultures
15%glycerol (375µl 60%glycerol + 1125µl cultute), 2 for each (different colonies):

  • attC/term/attC/mRFP
  • weak RBS/TetA(C)/LuxI/term
  • medium RBS/TetA(C)/LuxI/term
  • strong RBS/TetA(C)/LuxI/term
  • weak RBS/LacZ alpha/LuxI/term
  • strong RBS/LacZ alpha/LuxI/term



Minipreps of the yesterday's overnight cultures, 2 for each part(cultures starting from colonies #1 and #2 on each plate)
Elution in 35µl EB buffer

  • attC/term/attC/mRFP
  • weak RBS/TetA(C)/LuxI/term
  • medium RBS/TetA(C)/LuxI/term
  • strong RBS/TetA(C)/LuxI/term
  • weak RBS/LacZ alpha/LuxI/term
  • strong RBS/LacZ alpha/LuxI/term


Quantification of the DNA in the minipreps by Nanodrop

Sample name260/280260/230ng/µl
attC/term/attC/mRFP 11.97 2.11 159.6
attC/term/attC/mRFP 21.96 1.87 195.3
weakRBS/TetA(C)/LuxI/term 11.96 2.28381.3
weakRBS/TetA(C)/LuxI/term 21.96 2.30291.9
mediumRBS/TetA(C)/LuxI/term 11.96 2.26 353.2
mediumRBS/TetA(C)/LuxI/term 2 1.95 2.28490.1
standardRBS/TetA(C)/LuxI/term 1 1.962.27 425.2
standardRBS/TetA(C)/LuxI/term 21.96 2.31 362.6
weakRBS/LacZ alpha/LuxI/term 1 1.96 2.20 260.2
weakRBS/LacZ alpha/LuxI/term 2 1.97 2.28 273.3
standardRBS/LacZ alpha/LuxI/term 1 1.982.24 248.6
standardRBS/LacZ alpha/LuxI/term 2 1.97 2.23265.0


Restriction digests
Preparative digests (20µl miniprep + 2x1µl enzymes + 2.5µl buffer4 + 0.25µl BSA)
Minipreps from today (only with minipreps from colony #1):

  • attC/term/attC/mRFP with EcoRI-HF+XbaI
  • weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
  • medium RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
  • strong RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
  • weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI
  • strong RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI


Minipreps from 29/07:

  • pLux/GFP with EcoRI-HF+XbaI
  • Ps/weakRBS/LuxR with EcoRI-HF+SpeI
  • Pm/weakRBS/LuxR with EcoRI-HF+SpeI
  • Ps/mediumRBS/LuxR with EcoRI-HF+SpeI
  • Pm/mediumRBS/LuxR with EcoRI-HF+SpeI
  • Ps/stongRBS/LuxR with EcoRI-HF+SpeI
  • Pm/strongRBS/LuxR with EcoRI-HF+SpeI


Verification digests (5µl miniprep + 12µl water + 2x1µl enzymes + 2µl buffer4 + 0.2µl BSA)

  • attC/term/attC/mRFP (miniprep from colony #1) with EcoRI-HF+PstI
  • attC/term/attC/mRFP (miniprep from colony #2) with EcoRI-HF+PstI


Gel electrophoresis (1.5%agarose w/v, 50V)
20µl restriction digest+5µl 6x loading buffer weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI

  • weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
  • ladder
  • medium RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
  • -
  • strong RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI
  • -
  • weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI
  • -
  • strong RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI
  • -
  • attC/term/attC/mRFP #1 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #2 (digested with EcoRI-HF+PstI for verification)

Image:gel0108_1.jpg Image:gel0108_1cut.jpg


  • Ps/weakRBS/LuxR with EcoRI-HF+SpeI
  • -
  • Pm/weakRBS/LuxR with EcoRI-HF+SpeI
  • -
  • Ps/mediumRBS/LuxR with EcoRI-HF+SpeI
  • -
  • Pm/mediumRBS/LuxR with EcoRI-HF+SpeI
  • -
  • Ps/stongRBS/LuxR with EcoRI-HF+SpeI
  • -
  • Pm/strongRBS/LuxR with EcoRI-HF+SpeI
  • ladder


Image:Gel2_0108_cut_new.jpg‎ Image:gel0108_2cut.jpg


Gel extraction
Elution in 35µl warm water

  • weak RBS/TetA(C)/LuxI/term
  • medium RBS/TetA(C)/LuxI/term
  • strong RBS/TetA(C)/LuxI/term
  • weak RBS/LacZ alpha/LuxI/term
  • strong RBS/LacZ alpha/LuxI/term
  • Ps/weakRBS/LuxR
  • Pm/weakRBS/LuxR
  • Ps/mediumRBS/LuxR
  • Pm/mediumRBS/LuxR
  • Pm/strongRBS/LuxR


DNA quantification after gel extraction. Measured with the Nanodrop

Sample name260/280260/230ng/µl
weakRBS/TetA(C)/LuxI/term 11.77 0.13 27.1
mediumRBS/TetA(C)/LuxI/term 1 1.680.10 23.2
standardRBS/TetA(C)/LuxI/term 1 1.77 0.1627.3
weakRBS/LacZ alpha/LuxI/term 1 1.770.02 8.1
standardRBS/LacZ alpha/LuxI/term 1 1.590.03 9.3
Pm/standRBS/LuxR 1.900.01 3.5*
Pm/medRBS/LuxR 1.22 0.02 1.5*
Ps/medRBS/LuxR 2.200.03 2.5
Pm/weakRBS/LuxR 1.06 0.01 2.3*
Ps/weakRBS/LuxR 1.550.06 7.6


*Apparently no DNA in the sample : no peak on the Nanodrop.


PCR purification kit

  • pLux/GFP
  • attC/term/attC/mRFP (digested with EcoRI-HF and Xba-I for future ligation)


DNA quantification after PCR purification kit. Measured with the Nanodrop

Sample name260/280260/230ng/µl
attC/term/attC/mRFP (from colony#1)1.52 0.59 8.8
attC/term/attC/mRFP (from colony#2) 1.69 0.089.8
pLux/GFP (A from 29/07) 1.691.36 77.3



Ligation at 16°C overnight

  • 1µl pLux/GFP + 6µl stand.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase
  • 1µl pLux/GFP + 6µl med.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase
  • 1µl pLux/GFP + 6µl weak.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase
  • 1µl pLux/GFP + 16.5µl stand.RBS/LacZα/LuxI/term + 2µl buffer + 0.5µl ligase
  • 1µl pLux/GFP + 16.5µl weakRBS/LacZα/LuxI/term + 2µl buffer + 0.5µl ligase
  • control 1 : 1µl pLux/GFP + 1µl buffer + 7.5µl water + 0.5µl ligase
  • control 2 : 6µl stand.RBS/TetA(C)/LuxI/term + 1µl buffer + 2.5µl water + 0.5µl ligase


Overnight cultures

  • 12ml cultures LB+Kan started from 16 different colonies on the attC/term/attC/mRFP transformation plates from yesterday
  • 10ml culture LB+Kan of attC (from glycerol)
  • 10ml culture LB+Kan of term/attC/mRFP (from glycerol)


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