Team:INSA-Lyon/notebook

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Curly'on - IGEM 2014 INSA-LYON

IGEM

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


10/07

Digestion of pHC06 and pHC07 with N+P. Gel test showed digestion worked for pHC06 but not for pHC07.

Midi prep of Genecust constructions.

Determination of plasmids concentrations by nanodrop and by gel analysis.


11/07

Digestions of psB1C3, pHC06, pHC07 and the four DNA constructions with appropriate enzymes for ligation.

Electrophoresis gel test: the gel was too thin and DNA wasn’t enough concentrated so conclusions about digestions couldn’t be clearly drawn.

Ligations:

- 3A ligation of pHC06 with psB1C3 and each of the four Genecust constructions (csgA WT, His1, His2, mod)

- Standard ligation of pHC07 with each of the four DNA constructions (csgA WT, His1, His2, mod)

Transformation of DH5α strain with the assembled plasmids and selection on a Cm plate.


14/07

Extraction with mini prep ABC protocol of 120 clones in total of the transformed strains.


15/07

Simple digestion of all clones with EcoRI and verification by gel electrophoresis revealed no successful cloning.


16/07

Extraction with mini prep ABC of 4 new clones of pHC15 and pHC18 but and two clones seemed promising after verification with gel electrophoresis.

Dry lab: A new strategy for cloning was decided: gel purification of the inserts would be carried out after digestion, and assay test of phosphatase to eventually use it before ligation.

Digestions of pHC04 (from midi prep), pHC07 and 3 DNA constructions (Wild Type, His1X and His2X)

pHC07 was used to test phosphatase action : a control with 1,5 µL DNA and 8,5 µL water was made to see if some digestions were incomplete. Phosphatase was then inactivated 20 minutes at 80°C.

Verification with gel electrophoresis:

- Genecust constructions : successful digestion

- pHC04 : no digestion

- pHC07 : no digestion

Hypothesis to understand digestion failure: maybe one NEB 2 buffer was out of date.


17/07

Extraction with mini prep kit of these 2 clones: gels suggested that potentially good clone of pHC20 didn’t contain the plasmid and that the clone of pHC15 contained two vectors and the promoter but no Genecust construction.


25/06

Verification of buffers number 2 used for digestion: gel electrophoresis showed that problems of DNA digestions weren’t linked with buffer 2 as digestion worked with all buffers.

Ligation: 3A assembly of psB1C3, pHC06 and CsgA Wild Type (plasmid pHC14) from former digestions.

Verification by gel electrophoresis of:

- pHC14 (ligase + insert before and after ligation) : failure

- pHC07 (with and without digestion) : digestion of pHC07 seemed to have failed

- pHC09 (with and without digestion) : digestion seemed complete

Transformations in DH5 α and selection on Cm plates of:

- pHC14 (ligase + insert; ligase control and control) : hundreds of clones on “ligase + insert” plate against 10 more times on “ligase without insert” -> maybe due to an overproduction of CsgA (J23100 is a strong promoter) that kill bacteria ?

- pHC07 (with and without digestion) -> tests to check if the digestion was complete or not : confirmation of gel electrophoresis

- pHC09 (with and without digestion) -> tests to check if the digestion was complete or not : 7 clones after digestion against about 200 without digestion

Gel purification of pHC 10, 12 and 13: high amounts of DNA were obtained (even if there were some loss too) as the gel was overloaded.

Digestion of pHC11 (CsgA Modular) with XbaI and SpeI for future gel purification.

Digestion of pHC03 with BamHI and SpeI to test again the action of phosphatase enzyme.

Liquid preculture of pHC04 was prepared for next extraction with midi prep kit.


19/07

200mL liquid culture of pHC04 (dilution 1/1000 of preculture)


20/07


25/06


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