Modifications made on protocols allowed the proper conduct of the experiments even if we didn't get good results. $\backslash frac\; ab$

• Sequencing Order :
• RBS-CinI 2
• RBS-CinI 3
• RBS-CinI 5
• MerR-CinR
• MerR-CinR 4
• MerR-CinR 5
• RESULTS :
• MerR-CinR 5 came back positive from the sequencing
• Cloning Session (3A Assembly) :
• 1st-step constructions :
• RBS-TetR
• RBS-LuxR
• Fha-CinI
• Fha-CinR
• Fha-TetR
• Fha-LuxI
• Fha-LuxR

plasmid vector : pSB1AC3

• coloration constructions :
• pLux-Lycopene
• pCin-Lycopene

plasmid vector : pSB1AK3

• constructions for the tests :
• pConst-GFP
• pCin-GFP
• pLux-GFP
• pTet-GFP
• pLac-GFP

plasmid vector : pSB1AT3



• pConst-(RBS-CinR)
• pConst-(RBS-LuxR)
• plasmid vector: pSB3C5
• Digestions :

a gel checking of the restriction results were performed.

• Purification :

In order to check and extract the wright insert.

• Ligations :

with the ratio : 3X of insert 1X of plasmid

• Spreading over Petri dish
• Results :

No result on the PCR checking of the colonies.

• Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.
• Restriction :

Between E and P (same for pSB1AT3). No need of purification because PCR BlentII is Kanamycin resistant whereas the selection of pSB1AT3 is made on Tetracycline.

• Ligation
• Spreading over Petri Dish
• RESULTS :

PCR result: successful transfer.

• Cloning Session (Standard Assembly):
  
  • 1^st-step constructions:
  • RBS-TetR
  • RBS-LuxR
  • Fha-CinI
  • Fha-CinR
  • Fha-TetR
  • Fha-LuxI
  • Fha-LuxR

  Fha plasmid (pSB1AT3) is used as vector.

  • coloration constructions:
  • pLux-Lycopene
  • pCin-Lycopene

  Promoter plasmids are used as vector.

  • constructions for the tests:
  • pConst-GFP
  • pCin-GFP
  • pLux-GFP
  • pTet-GFP
  • pLac-GFP

  Promoter plasmids are used as vector.

  • pConst-(RBS-CinR)
  • pConst-(RBS-LuxR)

  Promoter plasmid is used as vector.

  • Digestions:

  a gel checking of the restriction results were performed.

  Purification: In order to check and extract the wright insert.

  Ligations

  Spreading over Petri Dish

  RESULTS :
  PCR result from colonies:
  5 potentially successfull constructions RBS-TetR Fha-CinI Fha-LuxI pLux-Lyco pCin-Lyco
  Double cheking with a restriction gel:
  from minpirep of the newly obtained constructions, we performed a digestion. Length of the interest fragment are then given by electroporesis
  -> same result as previously

  • Cloning Session :
    -> Constructions for the tests :
    3A Assembly
    pConst-(RBS-CinR) pConst-(RBS-LuxR)
    Plasmid vector: pSB3C5
    
    Standard Assembly
    pConst-GFP pLac-GFP pTet-GFP pLux-GFP pCin-GFP

  Digestions: a gel checking of the restriction results were performed.

  Purification: In order to check and extract the right insert.

  Ligations : We add a control for the ligations: plasmids without its inserts.

  Spreading over Petri dish

  RESULTS : Restriction gel checking:
  Only inserts from pConst-GFP and pLac-GFP constructions have the right size

  • Sequencing Order

  pConst-GFP Fha-LuxR Fha-LuxI Fha-CinI pLux-Lycopene pCin-Lycopene pLac-GFP RBS-TetR

  RESULTS : Correct sequences for pCin-Lycopene ad RBS-TetR