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Team:Grenoble/Notebook/August
From 2011.igem.org
August 4th to 10th
Biology
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
Biology
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
Biology
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.
Biology
Screening by enzymatic restriction (E and P) of constructs 19 and 21. Clone 19A,19B, 19E, 21A and 21B were sent to sequencing. Sequencing results shows expected constructs for all clones. Consequently they were stored at -80°C in 40% glycerol.
Screening of construction 22, 23 were performed first by colony PCR. Four clones for constructs 22 and 23 were retaineds for a restriction digest screening. Later experiments confirm colony PCR results for every clone tested.
They were all sent for sequencing.
Construction 24 was abandoned because two EcoRI restriction sites exist in pTOPO around 5’ and 3’ of blunt restriction sites (where standard rpoSLS was ligated). Construction with fhaLS-pTOPO (SpeII and Pst I) and I13401(XbaI and PstI) in standard assembly. fhaSL-I13401-pTOPO (construction 25) Construction 25 gave colonie but all contained empty plasmids as tested by colony PCR.
August 18th to 25th
Biology
Construction of construct 26 using fhaLS-pSB1AT3 constructed by (SpeI and Pst I) and I13401 (XhoI and PstI)
- fhaSL-I13401-pSB1AT2 (construct 26).
No colonies were obtained.
Sequencing results (clic to download pdf): construct 23 has the expected sequence. Concerning construct 22, the sequence is completely different. Construction 22 was abandoned.
Production of pSB1C3 by PCR and by minipreping. Insertion of rsmY from pTOPO into PSB1C3.
August 26th to 1st
Biology
Construction using standard assembly:
- fhaLS-I13401-pSB1AT2 (construction 28).
- R0040-rsmY-pSB1A2 (construction 29). RsmY were PCR products digested by XbaI and PstI. R0040 was digested by SpeI and PstI.
- J23119-magLS-I13401 (construction 30). With J23119, digested by SpeI and PstI, and construction 22, digested by XhoI and PstI.
Colony PCR and enzymatic restriction by XbaI and PstI was done for construction 29 and 30. Six clones per construction were sent to sequencing. Construction 28 failed (empty plasmids shown by colony PCR)
Insertion of rsmA in PSB1C3. Transformation of rsmA in PSB1C3 and rsmY in PSB1C3 by electroporation. Screen by PCR with VF and VR IGEM primers, using NEB Taq and appropriate protocol. Production of rposLS by PCR.
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