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Team:Grenoble/Notebook/August
From 2011.igem.org
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<li>Spreading over Petri dish</li> | <li>Spreading over Petri dish</li> | ||
<li>Results :</li> | <li>Results :</li> | ||
| - | < | + | <p>No result on the PCR checking of the colonies.</p> |
| - | + | ||
| - | + | ||
</ul> | </ul> | ||
| + | |||
<li>Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.</li> | <li>Plasmid transfer of Fha: from PCR BlentII to pSB1AT3.</li> | ||
<ul> | <ul> | ||
<li>Restriction :</li> | <li>Restriction :</li> | ||
<p> | <p> | ||
| - | Between E and P (same for pSB1AT3). No need of purification because PCR BlentII is Kanamycin resistant | + | 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. |
</p> | </p> | ||
<li>Ligation</li> | <li>Ligation</li> | ||
| Line 88: | Line 87: | ||
<p>PCR result: successful transfer.</p> | <p>PCR result: successful transfer.</p> | ||
</ul> | </ul> | ||
| - | |||
<li>Cloning Session (Standard Assembly):<br/> | <li>Cloning Session (Standard Assembly):<br/> | ||
| - | + | <ul> | |
| - | + | <li>1<sup>st</sup>-step constructions:</li> | |
| - | + | <ul> | |
| - | + | <li>RBS-TetR</li> | |
| - | -> | + | <li>RBS-LuxR</li> |
| + | <li>Fha-CinI</li> | ||
| + | <li>Fha-CinR</li> | ||
| + | <li>Fha-TetR</li> | ||
| + | <li>Fha-LuxI</li> | ||
| + | <li>Fha-LuxR</li> | ||
| + | <p>Fha plasmid (pSB1AT3) is used as vector.</p> | ||
| + | </ul> | ||
| + | <li>coloration constructions:</li> | ||
| + | </ul> | ||
| + | -> <br/> | ||
pLux-Lycopene pCin-Lycopene<br/> | pLux-Lycopene pCin-Lycopene<br/> | ||
Promoter plasmids are used as vector.<br/><br/> | Promoter plasmids are used as vector.<br/><br/> | ||
Revision as of 21:00, 20 September 2011
August 4th to 10th
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 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:
Fha plasmid (pSB1AT3) is used as vector.
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. - 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 - Sequencing Order
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.
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
August 11th to 17th
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 - Sequencing Order
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.
