IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/09/03

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Aleksandra and Raphaël
Testing of the counter on the plasmid (with mRFP) on the example of Ps/sRBS/LuxR/attC/term/attC/mRFP
Protocol : we took an overnight culture and diluted it 1/100 in 5 ml LB. When the OD was 0.4, we induced with Arabinose (0.1%, 0.2% and 0.4%). Then we plated every hour the 1/100 and 1/1000 dilutions on the plates without Ara.
The Ara serves to induce the expression of the integrase downstream of the pBAD/araC promoter on the pSWK plasmid. The integrase then recognises the attC sites on the pSB1A2 plasmid and excises the sequence between 2 sites : P/LuxR/attC/term/attC/mRFP → P/LuxR/mRFP. Thus, the terminator being excised the mRFP can be expressed downstream of the constitutive promoter. Red fluorescence appears only when Ara induced the integrase and it excised the terminator.
Here we have tested different ara pulses necessary for the integrase activation and action. We decided to use 0.1% ara induction for at least 4 hours, and we will be plating the 1/1000 dilutions.
Induction with 0.1% arabinose:
1h, 2h, 3h, 4h
Ara1 1.jpg Ara1 2.jpg Ara1 3.jpg Ara1 4.jpg
5h, 6h, 7h, 8h
Ara1 5.jpg Ara1 6.jpg Ara1 7.jpg Ara1 8.jpg


Induction with 0.2% arabinose:
1h, 2h, 3h, 4h
File:Ara2 1.jpg File:Ara2 2.jpg File:Ara2 3.jpg File:Ara2 4.jpg
5h, 6h, 7h, 8h
File:Ara2 5.jpg File:Ara2 6.jpg File:Ara2 7.jpg File:Ara2 8.jpg


Aleksandra
Minipreps

  • wLac
  • sLac
  • wTet
  • mTet
  • sTet
  • Ps


Restriction digest
with X+P :

  • wLac
  • sLac
  • wTet
  • mTet
  • sTet


with S+P

  • Ps


Gel electrophoresis

  • wTet
  • Ladder 1kb
  • mTet
  • -
  • sTet
  • -
  • sLac
  • -
  • wLac
  • -


File:Gel0309uncut.jpg
File:Gel0309cut.jpg


Gel extraction

  • wLac
  • sLac
  • wTet
  • mTet
  • sTet


PCR purif kit

  • Ps


Ligation

  • wLac + Ps
  • sLac + Ps
  • wTet + Ps
  • mTet + Ps
  • sTet + Ps
  • ctrl Ps


Overnight cultures started from 2 colonies per plate

  • attP/Pms/attC/RFP (in π)
  • attP/Psm/attC/RFP (in π)
  • attP/Pss/attC/RFP (in π)
  • Pms/mTet/LuxI
  • Pss/wTet/LuxI
  • Psm/sTet/LuxI
  • Psm/wTetIII/I13507/pLux/GFP
  • Psm/wTetII/I13507/pLux/GFP
  • Pss/sLac/LuxI III
  • Pms/sTet/LuxI