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

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.

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.

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

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

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

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)

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.