"
Team:Grenoble/Notebook/August
From 2011.igem.org
August 4th to 10th
BiologyMarion
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
- coloration constructions :
- pLux-Lycopene
- pCin-Lycopene
- constructions for the tests :
- pConst-GFP
- pCin-GFP
- pLux-GFP
- pTet-GFP
- pLac-GFP
- pConst-(RBS-CinR)
- pConst-(RBS-LuxR)
- plasmid vector: pSB3C5
- Digestions :
- Purification :
- Ligations :
- Spreading over Petri dish
- Results :
- Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.
- Restriction :
- Ligation
- Spreading over Petri Dish
- Results :
- Cloning Session (Standard Assembly):
- 1st-step constructions:
- RBS-TetR
- RBS-LuxR
- Fha-CinI
- Fha-CinR
- Fha-TetR
- Fha-LuxI
- Fha-LuxR
- coloration constructions:
- pLux-Lycopene
- pCin-Lycopene
- constructions for the tests:
- pConst-GFP
- pCin-GFP
- pLux-GFP
- pTet-GFP
- pLac-GFP
- pConst-(RBS-CinR)
- pConst-(RBS-LuxR)
- Digestions:
- PCR result from colonies :
- RBS-TetR
- Fha-CinI
- Fha-LuxI
- pLux-Lyco
- pCin-Lyco
- Double cheking with a restriction gel :
- same result as previously
Fha plasmid (pSB1AT3) is used as vector.
Promoter plasmids are used as vector.
Promoter plasmids are used as vector.
Promoter plasmid is used as vector.
a gel checking of the restriction results were performed.
Purification: In order to check and extract the wright insert.
Ligations
Spreading over Petri Dish
5 potentially successfull constructions:
From minpirep of the newly obtained constructions, we performed a digestion. Length of the interest fragment are then given by electroporesis.
- Cloning Session :
- Constructions for the tests (3A Assembly):
- pConst-(RBS-CinR)
- pConst-(RBS-LuxR)
- 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 :
- Sequencing Order
- pConst-GFP
- Fha-LuxR
- Fha-LuxI
- Fha-CinI
- pLux-Lycopene
- pCin-Lycopene
- pLac-GFP
- RBS-TetR
plasmid vector : pSB1AC3
plasmid vector : pSB1AK3
plasmid vector : pSB1AT3
a gel checking of the restriction results were performed.
In order to check and extract the wright insert.
with the ratio : 3X of insert 1X of plasmid
No result on the PCR checking of the colonies.
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.
PCR result: successful transfer.
Plasmid vector: pSB3C5
Restriction gel checking : Only inserts from pConst-GFP and pLac-GFP constructions have the right size
RESULTS : Correct sequences for pCin-Lycopene ad RBS-TetR
BiologyEric
Reception of two synthesized primers which are hybridized to obtain magLS. Construction from LS PCR product and linearized pSB1C3:
- rpoSLS-pSB1C3 (construction 19)
- magLS-pSB1C3 (construction 21)
Construction from LS PCR product and I13401 (standard assembly):
- fhaLS-I13401-pTOPO (construction 24)
- pSB1A2-rpoSLS-I13401 (construction 22)
- pSB1A2-magLS-I13401 (construction 23)
Colony PCR was performed for constructs 19 and 21 with primer VF and VR. Some clones were retained as positive. Digestion of retained clones by EcoRI (E) and PstI (P): Four clones of construct 19 and two of construct 21 were OK and sent to sequencing.
We try to set up a different PCR protocol to screen for the presence of an insert in pTOPO, using either two primers that hybridize on the plasmid, or one on the plasmid and one on the insert. None of the conditions we try work. We definitely give up with the use of this plasmid.
Having to use a new Taq (Pfu polymerase hight fidelity from Invitrogen) in a different lab, we set up the PCR for rposLS and rsmA again.
Production of those two sequences to be cloned into PSB1C3. Adaptation of the IGEM protocol for plasmid production to the Pfu polymerase.
August 11th to 17th
BiologyMarion
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 :
- 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
- pConst-GFP
- pTet-GFP
- pConst-(RBS-CinR)
- Sequencing Order :
- pLac-GFP
- pConst-GFP
- pTet-GFP
- pConst-(RBS-CinR)
- Fha in pSB1AT3
The DNA quantity added into the preparation was increased: 7µl instead of 2µl. A gel checking of the restriction results were performed.
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 :
Results : Sequences matched perfectly with theory for both inserts : pConst-GFP and Fha.
