Team:Lyon-INSA-ENS/Realisation/Week8

From 2011.igem.org

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Mesure de la DO600 des puits de la plaque poussant en LB/2, qui permet de calculer le pourcentage d'adhérence de chacune des souches.<br>
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Measurement of the OD600 of the LB/2 plate, which gives an adherence percentage for each strain.<br>
<br>
<br>
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Révélation de la plaque 24 puits en milieu M63. 1/4 des puits sont traités au Cristal Violet, et on prend la DO600 des autres.<br>
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Revealing of the M63 medium 24 well plate using the methyl violet ( 1 well) or OD600 method ( 3 wells ).
<br>
<br>
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Observation des tests de floculations lancés mardi. On observe de meilleurs résultats en milieu LB/2 par rapport au LB , bien qu'ils ne soient pas significatifs. On va donc garder le milieu LB/2 pour nos plaques 24 puits.<br>
 
<br>
<br>
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Mesure au nona drop des plasmides mis en collection.<br>
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The flocculation tests from the previous day give better results in LB/2 compared to LB , but they're not significative. We'll use LB/2 for future tests.<br>
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<br>
 +
Nano drop quantification of some plasmids in the collection.<br>
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On refait des plaques 24 puits, qui permettent de mettre en évidence :
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Start of new 24 well plates, to study:
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     <li> l'effet du Cobalt (en concentration constante), </il>
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     <li> the effect of cobalt (in increasing concentration), </il>
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     <li> la différence entre la souche Amp résistant et Cm résistant, </il>
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     <li> the difference between Amp or Cm strains, </il>
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     <li> l'effet de l'antibiotique,</il>
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     <li> the effect of the presence of antibiotic</il>
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     <li> l'effet du traitement de la plaque à l'EDTA,</li>
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     <li> the effect of prior treatment of the plate with EDTA</li>
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     <li> l'effet de l'EDTA (en concentration croissante) sur la souche. </il>
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     <li> the effect of EDTA (in increasing concentration) on the strain. </il>
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Caractérisation de OmpR234, avec une plaque 24 puits et un test de floculation<br>
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Characterization of OmpR234 with a 24 well plate and a flocculation test.<br>
<br>
<br>
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Test en milieu CFA qui permet de mettre en évidence la formation de Biofilm en le colorant en rouge.<br>
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Test in CFA medium to show the formation of biofilms by colouring them red using the following strains( 10µL each ) :<br>
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On met sur boîte 10 microL de PHL818 (témoin positif, souche adhérente), de NM522 (témoin négatif), de notre promoteur fort avec et sans la part OmpR234, de notre part de synthèse Amp résistant et celle Cm résistant.<br>
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PHL818 (positive control),NM522 (negative control), and test bacteria containing 18A promoter ( alone without the coding part ),our synthesized parts (Amp or Cm)<br>
<br>
<br>
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Construction de la part Prcn-GFP(LVA).<br>
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Construction of Prcn-GFP(LVA).<br>
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digestion du plasmide Prcn avec S et P = linéarisation du plasmide -> erreur : la part sort du plasmide <br>
+
Digestion of the plasmid with Prcn (S+P) = linearization -> error: the part goes out from the plasmid <br>
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-> utilisation d'autres tubes d'enzymes, mais mêmes résultats, idem pour la simple digestion -> nos tubes d'enzymes sont contaminés<br>
+
-> use of other enzyme tubes, with the same results, same with a simple digestion -> our enzyme tubes are contaminated<br>
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digestion plasmide RBS fort - GFP avec X et P -> OK<br>
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digestion of the plasmid with  strong RRB-GFP (X+P) -> OK<br>
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Répétition du test de floculation pour la caractérisation de OmpR234.<br>
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Repeat of the flocculation test for the characterization of OmpR234.<br>
<br>
<br>
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Répétition du test en milieu CFA.
+
Repeat of the CFA medium test.
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Revision as of 09:18, 16 August 2011









Week 8


From Monday the 1st of August to Friday the 5th of August 2011







Monday

24 well plate with LB/2 or M63 medium to test bacterial adherence in response to cobalt :
positive control with an adherent strain PHL818 ( MG1655 malT::Tn10 adr101=OmpR234 ),negative control with a non adherent strain NM522, and our synthesized part Rcn-CsgBAEFG (Amp) with increasing concentrations of cobalt ( in CoCl2 form).

Flocculation test for the same strains with LB/2 or M63 medium.

Miniprep of the transformed colonies with the following plasmids : Prcn / Pcurli-GFP / Pcurli - Operon curli.
-> none of the isolated clones had the Prcn or Pcurli-GFP plasmid, start of liquid culture of new clones.
-> two clones had the Pcurli-curli operon part : storage in the collection





Tuesday

In LB/2, the flocculation tests show flocculation for the adherent strain (PHL818), none for the negative control. Our strain shows a small flocculation that increases with the concentration in cobalt.
No flocculation in M63 medium for our strain : LB/2 will be used only for the next tests.

New flocculation tests with a wider range of cobalt concentration in LB or LB/2 and using the Rcn-CsgBAEFG (Cm) synthesized part

Revealing of the 24 well plate from the previous day, using 2 different methods : methyl violet (to visualise the formation of the biofilm at the surface of the well ) or direct OD600 measure.

Miniprep of the clones started the previous day
-> two clones have the Prcn part and one Pcurli-GFP.





Wednesday

Measurement of the OD600 of the LB/2 plate, which gives an adherence percentage for each strain.

Revealing of the M63 medium 24 well plate using the methyl violet ( 1 well) or OD600 method ( 3 wells ).

The flocculation tests from the previous day give better results in LB/2 compared to LB , but they're not significative. We'll use LB/2 for future tests.

Nano drop quantification of some plasmids in the collection.





Thursday

Start of new 24 well plates, to study:

  • the effect of cobalt (in increasing concentration),
  • the difference between Amp or Cm strains,
  • the effect of the presence of antibiotic
  • the effect of prior treatment of the plate with EDTA
  • the effect of EDTA (in increasing concentration) on the strain.

Characterization of OmpR234 with a 24 well plate and a flocculation test.

Test in CFA medium to show the formation of biofilms by colouring them red using the following strains( 10µL each ) :
PHL818 (positive control),NM522 (negative control), and test bacteria containing 18A promoter ( alone without the coding part ),our synthesized parts (Amp or Cm)

Construction of Prcn-GFP(LVA).
Digestion of the plasmid with Prcn (S+P) = linearization -> error: the part goes out from the plasmid
-> use of other enzyme tubes, with the same results, same with a simple digestion -> our enzyme tubes are contaminated
digestion of the plasmid with strong RRB-GFP (X+P) -> OK





Friday

Repeat of the flocculation test for the characterization of OmpR234.

Repeat of the CFA medium test.








ENS assystem Biomérieux INSA INSA
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