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Team:INSA-Lyon/notebook
From 2014.igem.org
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PROJECT
PROJECT- PROJECT SUMMARY
- ACHIEVEMENTS
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WETLAB
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MODELING
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HUMAN PRACTICE
HUMAN
PRACTICE- HUMAN PRACTICE SUMMARY
- SYNBIO PERCEPTION
- PROJECT'S INTEREST
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TEAM
NOTEBOOK
February-June
Literature searches were performed about Peptide Display, Curli biogenesis and metal trapping.
June
11/06
Reception of the four DNA constructions: CsgA Wild type, CsgA PolyHis1, CsgA PolyHis2, CsgA Modular
24/06
NM522 and DH5α cells (in liquid LB medium) were cultured overnight under agitation at 37°C for later transformation.
25/06
Transformation of NM522 and DH5α strains with each one of the following plasmids.
Selection of transformed bacteria on antibiotic plates depending on the plasmid.
26/06
Transformation analysis: all the transformations were successful except the ones with plasmids pHC03, pHC06, pHC07 and pHC09. Transformation of these four plasmids was repeated with a new protocol.
Clones of transformed strains containing pHC01, 02, 04, 05 and 08 were cultured in 5 mL LB medium containing 50 µL of the specific antibiotic at 37°C and appropriate plates were streaked with isolated colonies likely to contain transformed bacteria.
27/06
Transformation analysis: all the transformations were successful and plates containing pHC06 plasmids showed pink bacteria, as the rfp in the plasmid is expressed in presence of a strong promoter (like J23100).
A protocol for nickel titration was searched and nickel was stored for later experiments.
30/06
Extraction of the different plasmids with mini prep ABC protocol.
July
1/07
Transformation of the NM522 strain with the four constructions from Genecust
2/07
Digestion of all plasmids with restriction enzymes (except pHC05 which was incorrect, with one gene too many)
Electrophoresis gel test revealed no difference between the wells. Hypothesis: problems with enzymes or solutions A and C in mini prep ABC protocol.
A calibration curve of nickel was made using different concentrations of metal and DMG.
3/07
Strains containing the 9 plasmids were cultured in 5mL of LB medium + 50 µL specific antibiotic at 30°C.
Midi prep kit tested with pHC06. The kit was functional.
Gel test of pHC06 and nanodrop were both used to determine pHC06 concentration.
4/07
Storage of some strains such as ompR324 or csgA- that might be interesting for the project were put in storage.
New liquid cultures of the plasmids were incubated at 37°C this time.
5/07
Mini prep ABC solution tests: pHC06 was extracted with different combinations of solutions A and C and then digested. Solution C seemed to be the problem in previous ABC miniprep protocol.
Transformation of NM522 with pHC05.
8/07
Midi Prep of pHC01, pHC02, pHC04, pHC07, and pHC08.
Digestion of plasmids with appropriate restriction enzymes
Gel test results:
- pHC01 : no digestion
- pHC02 and 04 : digestion
- pHC07 : the simple digestion wasn’t enough to conclude
- pHC08 : 2 digestions with contradictory results
25/06
25/06
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