Just Team BeaverMarion

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 comeback positiv 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 hereas the selection of pSB1AT3 is made on Tetracycline.
Ligation
Spreading over Petri Dish


RESULTS :
PCR result: successful transfer.

• Cloning Session (Standard Assembly):
  -> 1st-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

Just Team BeaverMarion

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

Cloning Session (Standard Assembly):
-> 1st-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

-> 3rd-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.