IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/09

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Aleksandra
Minipreps, elution in 50µl warm water

  • Pst/weakRBS/LuxR
  • Pst/medRBS/LuxR
  • Pst/stRBS/LuxR
  • Pmed/weakRBS/LuxR
  • Pmed/medRBS/LuxR
  • Pmed/stRBS/LuxR
  • Pmed
  • Pst
  • K081014, colony2
  • I13507, colony2
  • I13507, colony3
  • pBAD/Int1 (Amp, glycerol45)
  • pBAD/Int1 (Kan, glycerol 18)
  • LuxI
  • pLux/RBS/GFP


Nanodrop

Sample name260/280 260/230 ng/µl
K081014, col2 1.942.13 222.8
I13507, col21.92 2.01 220.4
I13507, col3 1.96 1.99 187.7
pLux/RBS/GFP/term 1.91 2.09253.4
RBS/LuxI 1.93 1.85140.3
Pmed/weakRBS/LuxR 1.921.84 138.8
Pst/weakRBS/LuxR2.00 2.10117.3
Pmed/medRBS/LuxR1.93 2.09 210.6
Pstr/medRBS/LuxR 2.00 2.20153.5
Pmed/strRBS/LuxR1.86 1.76 122.8
Pstr/strRBS/LuxR 2.03 2.21145.6
Pmed 1.861.99 179.7
Pstr 1.98 2.06 181.4
Pbad/IntI (glycerol S45) 1.95 1.90 140.4
Pbad/IntI (glycerol S18) 2.02 2.35 152.4



Restriction digest

  • K081014 col2 with PstI and XbaI : 20µl miniprep + 2.5µl B3 + 0.25µl BSA + 2x1µl enzymes
  • I13507 col1 with PstI and XbaI : 20µl miniprep + 2.5µl B3 + 0.25µl BSA + 2x1µl enzymes
  • I13507 col2 with PstI and XbaI : 20µl miniprep + 2.5µl B3 + 0.25µl BSA + 2x1µl enzymes
  • LuxI with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • LuxI with XbaI and PstI : 5µl miniprep + 11µl water + 2µl B3 + 0.2µl BSA + 2x1µl enzymes
  • Ps with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pm with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • pBAD/Int1 (from glycerol45) with XbaI and PstI : 5µl miniprep + 11µl water + 2µl B3 + 0.2µl BSA + 2x1µl enzymes
  • pBAD/Int1 (from glycerol18) with XbaI and PstI : 5µl miniprep + 11µl water + 2µl B3 + 0.2µl BSA + 2x1µl enzymes



Raphaël
Restriction digest

  • K081014 with PstI and XbaI : 20µl miniprep + 2.5µl B3 + 0.25µl BSA + 2x1µl enzymes
  • Pst/weakRBS/LuxR/attC/term/attC/mRFP, 2 samples with SpeI and PstI : 15µl miniprep + 5µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pst/medRBS/LuxR/attC/term/attC/mRFP, 1 sample with SpeI and PstI : 15µl miniprep + 5µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pst/stRBS/LuxR/attC/term/attC/mRFP, 1 sample with SpeI and PstI : 15µl miniprep + 5µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pmed/weakRBS/LuxR/attC/term/attC/mRFP, 4 samples with SpeI and PstI : 15µl miniprep + 5µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pmed/stRBS/LuxR/attC/term/attC/mRFP, 3 samples with SpeI and PstI : 15µl miniprep + 5µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pst/weakRBS/LuxR/attC/term/attC/mRFP, 2 samples with EcoRI-HF and SpeI : 5µl miniprep + 15µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pst/medRBS/LuxR/attC/term/attC/mRFP, 1 sample with EcoRI-HF and SpeI : 5µl miniprep + 15µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pst/stRBS/LuxR/attC/term/attC/mRFP, 1 sample with EcoRI-HF and SpeI : 5µl miniprep + 15µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pmed/weakRBS/LuxR/attC/term/attC/mRFP, 4 samples with EcoRI-HF and SpeI : 5µl miniprep + 15µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes
  • Pmed/stRBS/LuxR/attC/term/attC/mRFP, 3 samples with EcoRI-HF and SpeI : 5µl miniprep + 15µl water + 2.5µl B2 + 0.25µl BSA + 2x1µl enzymes


Léa
Restriction digest

  • RBS/LuxI/term (TURBOcell plate 1, col1) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzyme + 2.5µl B4 + 0.25µl BSA
  • RBS/LuxI/term (TURBOcell plate 2, col1) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzyme + 2.5µl B4 + 0.25µl BSA
  • RBS/LuxI/term (TURBOcell plate 2, col2) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzyme + 2.5µl B4 + 0.25µl BSA
  • RBS/LuxI/term (TOP10, col1) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzyme + 2.5µl B4 + 0.25µl BSA
  • RBS/LuxI/term (TOP10, col2) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzyme + 2.5µl B4 + 0.25µl BSA
  • weakRBS/TetA(C) with XbaI and PstI : 20µl miniprep + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA
  • medRBS/TetA(C) with XbaI and PstI : 20µl miniprep + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA
  • strRBS/TetA(C) with XbaI and PstI : 20µl miniprep + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA
  • weakRBS/LacZα with XbaI and PstI : 20µl miniprep + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA
  • strRBS/LacZα with XbaI and PstI : 20µl miniprep + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA


Gel electrophoresis
Gel 1

  • K081014
  • -
  • Pst/stRBS/LuxR/attC/term/attC/mRFP, 1 sample with SpeI and PstI
  • -
  • Pmed/weakRBS/LuxR/attC/term/attC/mRFP, 4 samples with SpeI and PstI
  • Ladder 100bp
  • Pst/weakRBS/LuxR/attC/term/attC/mRFP, 2 samples with SpeI and PstI
  • Ladder 1kb

Image:gel0809_1.jpg


Gel 2

  • Pst/medRBS/LuxR/attC/term/attC/mRFP, 1 sample with SpeI and PstI
  • -
  • Pmed/stRBS/LuxR/attC/term/attC/mRFP,3 samples with SpeI and PstI
  • -
  • weakRBS/TetA(C)
  • -
  • medRBS/TetA(C)
  • -
  • strRBS/TetA(C)
  • -

Image:gel0809_2.jpg


Gel 3

  • Pst/weakRBS/LuxR/attC/term/attC/mRFP, 2 samples with EcoRI-HF and SpeI
  • Pst/medRBS/LuxR/attC/term/attC/mRFP, 1 sample with EcoRI-HF and SpeI
  • Pst/stRBS/LuxR/attC/term/attC/mRFP, 1 sample with EcoRI-HF and SpeI
  • Pmed/weakRBS/LuxR/attC/term/attC/mRFP, 4 samples with EcoRI-HF and SpeI
  • Pmed/stRBS/LuxR/attC/term/attC/mRFP, 3 samples with EcoRI-HF and SpeI
  • Ladder 1kb

Image:gel0809_3.jpg


Gel 4

  • strRBS/LacZα
  • Ladder 100bp
  • weakRBS/LacZα
  • Ladder 1kb
  • pBAD/IntI (from glycerol S45)
  • pBAD/IntI (from glycerol S18)
  • RBS/LuxI/term (TURBOcell plate 1, col1)
  • RBS/LuxI/term (TURBOcell plate 2, col1)
  • RBS/LuxI/term (TURBOcell plate 2, col2)
  • RBS/LuxI/term (TOP10, col1)
  • RBS/LuxI/term (TOP10, col2)

Image:gel0809_4.jpg

Raphaël and Aleksandra
Starting overnight cultures from glycerols

  • 11 attC/term/attC/mRFP
  • Pst/weakRBS/LuxR
  • Pst/medRBS/LuxR
  • Pst/stRBS/LuxR
  • Pmed/weakRBS/LuxR
  • Pmed/medRBS/LuxR
  • Pmed/stRBS/LuxR
  • Pmed
  • Pst
  • wRBS/TetA(C)
  • medRBS/TetA(C)
  • stRBS/TetA(C)
  • wRBS/LacZα
  • stRBS/LacZα


Analyzing the sequencing results All of the following sequences are correct :

  • Pst/weakRBS/LuxR
  • Pst/medRBS/LuxR
  • Pst/stRBS/LuxR
  • Pmed/weakRBS/LuxR
  • Pmed/medRBS/LuxR
  • Pmed/stRBS/LuxR


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