Modifications made on protocols allowed the proper conduct of the experiments even if we didn't get good results.

• 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

  • 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

Few manipulation this week, but we also worked on the tests of our project for the Human Practice.

• Cloning Session (Standard Assembly):
• 1^st-step constructions:
• RBS-CinI
• RBS-LuxI
• RBS-LacI
• Fha-CinR
• Fha-LacI
• Constructions for the tests:
• pCin-GFP
• pLux-GFP
• pLac-GFP
• pConst-GFP
• pConst-(RBS-CinR)
• pConst-MerR-CinR
• 3^rd-step Construction:
• pLac-MerR-CinR
• Digestions :

The DNA quantity added into the preparation was increased: 7µl instead of 2µl. A gel checking of the restriction results were performed.

• Purification : In order to check and extract the wright insert.
• Ligations : We add a control for the ligations: plasmids without its inserts.
• Spreading over Petri dish

Results : Owing to an antibiotic issue, bacterial mats were obtained on every Petri Dishes. So, remaining preculture were spread again. And no bacteria growd this time. Ligations were started over using purified DNA from the previous digestions : PCR checking were performed on colonies, just few inserts had the right length :

• pConst-GFP
• pTet-GFP
• pConst-(RBS-CinR)
• Sequencing Order :
• pLac-GFP
• pConst-GFP
• pTet-GFP
• pConst-(RBS-CinR)
• Fha in pSB1AT3

Results : Sequences matched perfectly with theory for both inserts : pConst-GFP and Fha.