Team:Lyon-INSA-ENS/Realisation/Week8

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     <h1 style="color: white;"> Week 8 </h1>     
     <h1 style="color: white;"> Week 8 </h1>     
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   <br/>
   <br/>
-
<p style=" line-height : 1.5em">
 
-
24 well plate with LB/2 or M63 medium to test bacterial adherence in response to cobalt :<br>
 
-
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).<br>
 
 +
 +
 +
<p style=" line-height : 1.5em; ">
 +
<FONT COLOR="purple"><b> Adherence and Flocculation Tests</b></FONT> <br><br>
 +
<b>24 well plate</b> with LB/2 or M63 medium to test bacterial adherence in response to cobalt :<br>
 +
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).<br>
 +
</p>
 +
 +
<p style=" line-height : 1.5em;">
<br>
<br>
-
Flocculation test for the same strains with LB/2 or M63 medium.<br>
+
<b>Flocculation test</b> for the same strains with LB/2 or M63G medium.<br>
-
<br>
+
<br><br/>
-
Miniprep of the transformed colonies with the following plasmids : Prcn / Pcurli-GFP / Pcurli - Operon curli.<br>
+
 
 +
</p>
 +
<p style=" line-height : 1.5em;"><FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
 +
 
 +
<b>Miniprep</b> of the transformed colonies with the following plasmids : Prcn / Pcurli-GFP / Pcurli - Operon curli.<br>
-> none of the isolated clones had the Prcn or Pcurli-GFP plasmid, start of liquid culture of new clones.<br>
-> none of the isolated clones had the Prcn or Pcurli-GFP plasmid, start of liquid culture of new clones.<br>
-> two clones had the Pcurli-curli operon part : storage in the collection <br>
-> two clones had the Pcurli-curli operon part : storage in the collection <br>
Line 53: Line 69:
     <br/>
     <br/>
     <p style=" line-height : 1.5em">
     <p style=" line-height : 1.5em">
 +
<FONT COLOR="purple"><b> Adherence and Flocculation Tests</b></FONT> <br><br>
 +
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.<br>
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.<br>
No flocculation in M63 medium for our strain : LB/2 will be used only for the next tests. <br>
No flocculation in M63 medium for our strain : LB/2 will be used only for the next tests. <br>
<br>
<br>
-
New flocculation tests with a wider range of cobalt concentration in LB or LB/2 and using the Rcn-CsgBAEFG (Cm) synthesized part <br>
+
New <b>flocculation tests</b> with a wider range of cobalt concentration in LB or LB/2 and using the Prcn-CsgBAEFG (Cm) synthesized part <br>
 +
    </p>
 +
 
 +
<div style="border:1px solid black;float:right;margin-right:5%; width : 210px">
 +
    <img src="https://static.igem.org/mediawiki/2011/f/f5/02.08_24_well_plate.jpg" width="210px"/>   
 +
</div>
 +
 
 +
<p style=" line-height : 1.5em; width : 400px">
<br>
<br>
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.<br>
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.<br>
-
<br>
+
  </p>
-
Miniprep of the clones started the previous day <br>
+
<br><br/>
 +
 
 +
    <p style=" line-height : 1.5em">
 +
 
 +
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
 +
 
 +
<b>Miniprep</b> of the clones started the previous day <br>
-> two clones have the Prcn part and one Pcurli-GFP.<br>  
-> two clones have the Prcn part and one Pcurli-GFP.<br>  
 +
<br>
 +
<b>Storage</b> of PHL818, PHL1273, Rcn-CsgBAEFG (Cm) = S3 = S17, Rcn-CsgBAEFG (Amp) = S4 = S18<br>
   </p>   
   </p>   
Line 69: Line 102:
<h6 style="text-align :left"> Wednesday </h6><HR>
<h6 style="text-align :left"> Wednesday </h6><HR>
-
    <br/>
+
 
 +
<br>
-
    <p style=" line-height : 1.5em">
+
<div style="border:1px solid black;float:right;margin-right:5%; width : 180px">
-
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>
+
    <img src="https://static.igem.org/mediawiki/2011/9/92/03.08_24_well_plate.jpg" width="180px"/>   
 +
</div>
 +
 
 +
      <p style=" line-height : 1.5em ; width : 440px">
 +
<FONT COLOR="purple"><b> Adherence and Flocculation Tests</b></FONT> <br><br>
 +
Measurement of the OD600 of the LB/2 plate, which gives an adherence percentage for each strain.
 +
  <br/> <br/>
 +
Revealing of the M63 medium 24 well plate using the methyl violet ( 1 well) or OD600 method ( 3 wells ).
 +
<br>  <br/>
 +
      </p>
 +
 
 +
  <p style=" line-height : 1.5em">
<br>
<br>
-
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>
+
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><br/>
-
<br>
+
 
-
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>
+
<div style="border:1px solid black;float:left;margin-left:5%; width : 448px">
-
<br>
+
    <img src="https://static.igem.org/mediawiki/2011/4/46/03.08_LB_grand_test_floc.jpg" />
-
Mesure au nona drop des plasmides mis en collection.<br>
+
</div>
 +
 
 +
<p style=" line-height : 1.5em; width : 200px ; margin-left : 70%">
 +
<br><br/><br/>
 +
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/><br/><br/><br/> <br/><br/><br/><br/>
 +
</p>
 +
 
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="blue"><b>Plasmid Collection</b></FONT> <br><br>
 +
<b>Nanodrop</b> quantification of some plasmids in the collection.<br><br><br>
     </p>   
     </p>   
 +
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR=#12B1AF><b>Cobalt accumulating strain reconstruction</b></FONT> <br><br>
 +
MC4100 E.coli put in LB medium, with 10mM MgSO4 and 10mM CaCl2<br>
 +
 +
    </p> 
 +
     <br/> <br/>
     <br/> <br/>
Line 89: Line 151:
     <br/>
     <br/>
  <p style=" line-height : 1.5em">
  <p style=" line-height : 1.5em">
-
On refait des plaques 24 puits, qui permettent de mettre en évidence :
+
<FONT COLOR="purple"><b> Tests</b></FONT> <br><br>
 +
Start of new <b>24 well plates</b>, to study:
   </p>
   </p>
<ul style=" line-height : 1.5em">
<ul style=" line-height : 1.5em">
-
     <li> l'effet du Cobalt (en concentration constante), </il>
+
     <li> the effect of cobalt (in increasing concentration), </il>
-
     <li> la différence entre la souche Amp résistant et Cm résistant, </il>
+
     <li> the difference between Amp or Cm strains, -> it works better in Amp </il>
-
     <li> l'effet de l'antibiotique,</il>
+
     <li> the effect of the presence of antibiotic, ->  it works better with Antibiotic</il>
-
     <li> l'effet du traitement de la plaque à l'EDTA,</li>
+
     <li> the effect of prior treatment of the plate with EDTA, -> it is better when plate is rinse</li>
-
     <li> l'effet de l'EDTA (en concentration croissante) sur la souche. </il>
+
     <li> the effect of EDTA (in increasing concentration) on the strain. -> no useful </il>
</ul>
</ul>
<br>
<br>
<p style=" line-height : 1.5em">
<p style=" line-height : 1.5em">
-
Caractérisation de OmpR234, avec une plaque 24 puits et un test de floculation<br>
+
Characterization of OmpR234 with a <b>24 well plate</b> and a <b>flocculation test</b>.<br>
<br>
<br>
-
Test en milieu CFA qui permet de mettre en évidence la formation de Biofilm en le colorant en rouge.<br>
+
</p>
-
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>
+
 
-
<br>
+
<div style="float:left;margin-left:5%; width : 170px">
-
Construction de la part Prcn-GFP(LVA).<br>
+
    <img src="https://static.igem.org/mediawiki/2011/8/89/05.08_test_CFA.jpg" width="170px"/>   
-
digestion du plasmide Prcn avec S et P = linéarisation du plasmide -> erreur : la part sort du plasmide <br>
+
</div>
-
-> utilisation d'autres tubes d'enzymes, mais mêmes résultats, idem pour la simple digestion -> nos tubes d'enzymes sont contaminés<br>
+
 
-
digestion plasmide RBS fort - GFP avec X et P -> OK<br>
+
<p style=" line-height : 1.5em; margin-left: 35%">
 +
<br/> <br/>
 +
<b>Test in CFA medium</b> to show the formation of biofilms by colouring them red using the following strains( 10µL each ) :<br>
 +
PHL818 (positive control),NM522 (negative control), and test bacteria containing 18A promoter ( alone without the coding part ),our synthesized parts (Amp or Cm)<br>
 +
-> not very conclusive, a little circle of light around the bacteria.
 +
<br><br/> <br/><br/>
 +
</p>
 +
 
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="red"> <b>Strain construction : Prcn-GFP(LVA)</b> </FONT> <br><br>
 +
 
 +
<b>Digestion</b> of the plasmid with Prcn (S+P) = linearization -> error: the part goes out from the plasmid <br>
 +
-> use of other enzyme tubes, with the same results, same with a simple digestion -> our enzyme tubes are contaminated<br>
 +
digestion of the plasmid with  strong RRB-GFP (X+P) -> OK<br>
 +
<br><br/>
 +
 
 +
</p>
 +
<p style=" line-height : 1.5em"><FONT COLOR="blue"><b>Strain Collection</b></FONT> <br><br>
 +
<b>Storage</b> of MC4100 (Str) = S7, NM522 = S11, NMYO = S13 and NM522/NicoT (Amp) = S14
 +
    </p><br/><br/><br/>
 +
 
 +
 
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR=#12B1AF><b>Cobalt accumulating strain reconstruction</b></FONT> <br><br>
 +
Created a dilution range for the P1 phage we've been given (10-2 to 10-10)<br>
 +
Phage adsorbption onto our target strain <br/>
 +
Overnight culture, 37°C <br/>
     </p>   
     </p>   
Line 114: Line 203:
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
 +
  <h6 style="text-align :left"> Friday</h6><HR>
  <h6 style="text-align :left"> Friday</h6><HR>
   <br>
   <br>
 +
  <p style=" line-height : 1.5em">
  <p style=" line-height : 1.5em">
-
Répétition du test de floculation pour la caractérisation de OmpR234.<br>
+
<FONT COLOR="purple"><b> Tests</b></FONT> <br><br/>
 +
Repeat of the <b>flocculation test</b> for the characterization of OmpR234.<br>
<br>
<br>
-
Répétition du test en milieu CFA.
+
Repeat of the <b>CFA medium test</b>.<br>
 +
<br>
 +
Revealing of the four 24 well plates from thursday.
 +
    </p> 
 +
 
 +
 
 +
<br/><br/><br/><p style=" line-height : 1.5em">
 +
<FONT COLOR=#12B1AF><b>Cobalt accumulating strain reconstruction</b></FONT> <br><br>
 +
Revealing of the lysis plates : results are negative, it seems the phage are inactive. Re-construction post-poned indefinitely.<br/>
 +
 
     </p>   
     </p>   
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
-
 
+
    <br/> <br/>
 +
    <p>
 +
              <a  href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week7"/><font color="grey"><b>Previous Week</b></font></a>
 +
              <a  style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week9"/><font color="grey"><b>Next Week</b></font></a>
 +
              <br/>
 +
    </p>
 +
    <br/> <br/>
</div>
</div>

Latest revision as of 23:35, 21 September 2011







Week 8


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







Monday

Adherence and Flocculation Tests

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 M63G medium.


Strain construction

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

Adherence and Flocculation Tests

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 Prcn-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.



Strain construction

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

Storage of PHL818, PHL1273, Rcn-CsgBAEFG (Cm) = S3 = S17, Rcn-CsgBAEFG (Amp) = S4 = S18





Wednesday

Adherence and Flocculation Tests

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.




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.







Plasmid Collection

Nanodrop quantification of some plasmids in the collection.


Cobalt accumulating strain reconstruction

MC4100 E.coli put in LB medium, with 10mM MgSO4 and 10mM CaCl2





Thursday

Tests

Start of new 24 well plates, to study:

  • the effect of cobalt (in increasing concentration),
  • the difference between Amp or Cm strains, -> it works better in Amp
  • the effect of the presence of antibiotic, -> it works better with Antibiotic
  • the effect of prior treatment of the plate with EDTA, -> it is better when plate is rinse
  • the effect of EDTA (in increasing concentration) on the strain. -> no useful

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)
-> not very conclusive, a little circle of light around the bacteria.



Strain construction : 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


Strain Collection

Storage of MC4100 (Str) = S7, NM522 = S11, NMYO = S13 and NM522/NicoT (Amp) = S14




Cobalt accumulating strain reconstruction

Created a dilution range for the P1 phage we've been given (10-2 to 10-10)
Phage adsorbption onto our target strain
Overnight culture, 37°C





Friday

Tests

Repeat of the flocculation test for the characterization of OmpR234.

Repeat of the CFA medium test.

Revealing of the four 24 well plates from thursday.




Cobalt accumulating strain reconstruction

Revealing of the lysis plates : results are negative, it seems the phage are inactive. Re-construction post-poned indefinitely.







Previous Week Next Week






ENS assystem Biomérieux INSA INSA
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