IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/14

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Aleksandra
Minipreps from 10ml culture, elution in 35µl warm water

  • attP HK wt col1
  • attP HK wt col2
  • attP HK mut col1
  • attP HK mut col2
  • attP λ mut col1
  • attP λ mut col2


Nanodrop of the minipreps

Sample name 260/280 260/230 ng/µl
attP HK wt col1 1.962.35 340.1
attP HK wt col2 1.922.32 411.5
attP HK mut col11,92 2.29 329.1
attP HK mut col2 1.97 2.30 282.7
attP λ mut col1 1.952.24 282.7
attP λ mut col2 1.93 2.21 264.0
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Aleksandra
Gel extraction, elution in 35µl warm water

  • Ps/sRBS/LuxR/attC/term/attC
  • Pm/sRBS/LuxR/attC/term/attc
  • Ps/mRBS/LuxR/attC/term/attc
  • Ps/wRBS/LuxR/attC/term/attc
  • RBS/LuxI


PCR kit purification, elution in 35µl warm water

  • wRBS/LacZα
  • stRBS/lacZα
  • pLux
  • Pm/wRBS/LuxR
  • Pm/mRBS/LuxR


Nanodrop

Sample name 260/280 260/230 ng/µl
Ps/sRBS/LuxR/attC/term/attC 1.860.18 63.2
Pm/sRBS/LuxR/attC/term/attc 1.82 0.36 75.2
Ps/mRBS/LuxR/attC/term/attc 1.74 0.43 27.8
Ps/wRBS/LuxR/attC/term/attc 1.83 0.42 33.3
RBS/LuxI 1.67 0.03 8.2
wRBS/LacZα1.83 1.99 28.3
stRBS/lacZα 1.771.18 42.7
pLux 1.76 1.5156.1
Pm/wRBS/LuxR 1.560.76 42.5
Pm/mRBS/LuxR 1.751.76 35.5



Ligation for 45mins at 25°C

  • pLux+LuxI : 1µl + 18µl + 2.2 buffer + 0.5µl ligase
  • Pm/wRBS+attC/t/attC/mRFP : 0.5µl + 5µl + 0.5µl boffer + 0.5µl ligase
  • Pm/mRBS+attC/t/attC/mRFP : 0.5µl + 5µl + 0.5µl boffer + 0.5µl ligase

Controls :

  • pLux : 1µl + 18µl water + 2.2 buffer + 0.5µl ligase
  • Pm/wRBS : 0.5µl + 5µl water + 0.5µl boffer + 0.5µl ligase
  • Pm/mRBS : 0.5µl + 5µl water + 0.5µl boffer + 0.5µl ligase


Transformation of DH5α cells prepares by last year's iGEM team (P1)
With today's ligations:

  • pLux+LuxI
  • Pm/wRBS+attC/t/attC/mRFP
  • Pm/mRBS+attC/t/attC/mRFP

Controls :

  • pLux
  • Pm/wRBS
  • Pm/mRBS
  • positive control (pUC19)


Double transformation to verify the phenotype (RFP) when the integrase is activated

  • pSUlib (pBAD/IntI, Kan) and pSB1A2 (Ps/sRBS/attC/term/attC/mRFP, Amp)
  • pSUlib (pBAD/IntI, Kan) and pSB1A2 (Ps/mRBS/attC/term/attC/mRFP, Amp)
  • pSUlib (pBAD/IntI, Kan) and pSB1A2 (Ps/wRBS/attC/term/attC/mRFP, Amp)
  • pSUlib (pBAD/IntI, Kan) and pSB1A2 (Pm/sRBS/attC/term/attC/mRFP, Amp)



Overnight cultures

  • wRBS/TetA(C)
  • mRBS/TetA(C)
  • stRBS/TetA(C)
  • wRBS/LacZα
  • sRBS/LacZα
  • RBS/LuxI


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