Team:INSA-Lyon/notebook

From 2014.igem.org

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<p> NM522 and DH5α cells (in liquid LB medium) were cultured overnight under agitation at 37°C for later transformation. </p>
<p> NM522 and DH5α cells (in liquid LB medium) were cultured overnight under agitation at 37°C for later transformation. </p>
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<p>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). </p>
<p>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). </p>
<p>A protocol for nickel titration was searched and nickel was stored for later experiments. </p>
<p>A protocol for nickel titration was searched and nickel was stored for later experiments. </p>
 +
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<br/><h6>30/06 </h6>
 +
<br/>
 +
<p> Extraction of the different plasmids with mini prep ABC protocol.</p>
 +
<br/>
 +
 +
<br/>
 +
<h5>July </h5>
 +
<br/>
 +
 +
<h6>1/07 </h6>
 +
<br/>
 +
<p>Transformation of the NM522 strain with the four constructions from Genecust </p>
 +
<br/>
 +
 +
<h6>2/07 </h6>
 +
<br/>
 +
<p> Digestion of all plasmids with restriction enzymes (except pHC05 which was incorrect, with one gene too many)</p>
 +
<p> Electrophoresis gel test revealed no difference between the wells. Hypothesis: problems with enzymes or solutions A and C in mini prep ABC protocol.
 +
</p>
 +
<p>A calibration curve of nickel was made using different concentrations of metal and DMG.</p>
 +
<br/>
 +
 +
<h6>3/07 </h6>
 +
<br/>
 +
<p>Strains containing the 9 plasmids were cultured in 5mL of LB medium + 50 µL specific antibiotic at 30°C.  </p>
 +
<p>Midi prep kit tested with pHC06. The kit was functional.  </p>
 +
<p>Gel test of pHC06 and nanodrop were both used to determine pHC06 concentration. </p>
 +
<br/>
 +
 +
<h6>4/07 </h6>
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<br/>
 +
<p> Storage of some strains such as ompR324 or csgA- that might be interesting for the project were put in storage. </p>
 +
<p> New liquid cultures of the plasmids were incubated at 37°C this time. </p>
 +
<br/>
 +
 +
<h6>5/07 </h6>
 +
<br/>
 +
<p> 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.</p>
 +
<p> Transformation of NM522 with pHC05.</p>
 +
 +
<br/><h6>8/07 </h6>
 +
<br/>
 +
<p> Midi Prep of pHC01, pHC02, pHC04, pHC07, and pHC08.</p>
 +
<p> Digestion of plasmids with appropriate restriction enzymes </p>
 +
<p> Gel test results: </p>
 +
<p> - pHC01 : no digestion</p>
 +
<p> - pHC02 and 04 : digestion </p>
 +
<p> - pHC07 : the simple digestion wasn’t enough to conclude</p>
 +
<p> - pHC08 : 2 digestions with contradictory results</p>
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<br/>
 +
 +
<h6>25/06 </h6>
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Revision as of 22:53, 10 October 2014

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


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


25/06


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