Team:Lyon-INSA-ENS/Realisation/Week13

From 2011.igem.org

(Difference between revisions)
 
(20 intermediate revisions not shown)
Line 7: Line 7:
<html>
<html>
 +
 +
<div style="float : left; margin-top : -10px; margin-left : -200px">
 +
  <a href="https://2011.igem.org/Main_Page" >
 +
      <img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" />
 +
  </a>
 +
</div>
<body>
<body>
     <div id="language";>
     <div id="language";>
-
         <img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Realisation/Week13Fr">Version Francaise</a>
+
         <img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Realisation/Week13Fr">Version Fran&ccedil;aise</a>
     </div>
     </div>
Line 17: Line 23:
     <br/> <br/>
     <br/> <br/>
-
     <br/> <br/>
+
     <br/>  
-
    <br>
+
-
<div class="cadre" ; style="background-color:green;" >
+
<div class="cadre" ; style="background-color:green;margin-left : 8%" >
     <br/>
     <br/>
     <h1 style="color: white;"> Week 13 </h1>     
     <h1 style="color: white;"> Week 13 </h1>     
Line 26: Line 31:
<br/>
<br/>
-
     <p style="text-align : center"> <small> From Monday the 12th of September to Friday the 16th of September 2011 </small> </p>
+
     <p style="text-align : center"> <small> From Monday the 12th of September to Sunday the 18th of September 2011 </small> </p>
     <br/> <br/>
     <br/> <br/>
Line 34: Line 39:
   <h6 style="text-align :left"> Monday </h6> <HR>
   <h6 style="text-align :left"> Monday </h6> <HR>
    
    
-
   <br/>
+
   <br/><br/>
  <p style=" line-height : 1.5em">
  <p style=" line-height : 1.5em">
-
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
+
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/>
 +
Extraction of PHL1414+pIG3, PHL1414+pIG16 (clone 1,2,3) , MC4100+piG34 200µl (clone 1,2), MC4100+pIG34 800µl (clone 1, 2).<br/><br/>
 +
 +
Digestion by EcoRI and PstI.<br/><br/>
 +
 +
Gel electrophoresis to verify the ligations. Inserts have the good length for pIG3, pIG16 so the transformation went well.
 +
Inserts of pIG34 200µl clone 1 and pIG34 800µl clone 1 are good so the ligation went well. We’re keeping in collection the pIG34 200µl clone 1 as pIG34 in the plasmids’ collection and MC400+pIG34 as S32 in the strains’ collection.<br/><br/>
 +
 +
Cloning and transformation of rcn-ompR234 + pSB1C3 in MC4100.<br/><br/><br/>
</p>
</p>
Line 44: Line 57:
<p style=" line-height : 1.5em">
<p style=" line-height : 1.5em">
<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/>
<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/>
-
Plating of S17 from the collection to extract a bigger quantity of pIG25 ( pSB1C3 containing rcn-csgBAEFG )<br/><br/>
+
Plating of S17 from the collection to extract a bigger quantity of pIG25 (pSB1C3 containing rcn-csgBAEFG).<br/><br/>
Storage of the correct clone of:<br/>
Storage of the correct clone of:<br/>
Line 51: Line 64:
MC4100 + pIG34 (S32) <br/><br/>
MC4100 + pIG34 (S32) <br/><br/>
-
Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep.
+
Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep. <br/><br/><br/>
</p>
</p>
 +
 +
<p style=" line-height : 1.5em">
<p style=" line-height : 1.5em">
-
<FONT COLOR="purple"><b> Adherence Test Preparation </b></FONT> <br/><br/>
+
<FONT COLOR="purple"> <b> Microscopy Test </b> </FONT> <br/><br/>
 +
 
 +
<b>Preparation</b><br/>
 +
Sterilization of microscope cover slips in ethanol during a few minutes, then rinsed in sterilized water. <br/><br/>
 +
 
 +
<b>Test</b> <br/>
 +
 
 +
Preparation of plates for the following strains (one repetition):<br/>
 +
-PHL1414/piG3 AmpR = negative control without Co and with Co 25µM<br/>
 +
-PHL1414/piG16 AmpR without Co, Co 10µM, Co 25µM, Co 50µM and Co 100µM<br/><br/>
 +
 
 +
Incubation at 30°C overnight (during 16 hours).<br/><br/><br/>
Line 66: Line 92:
Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing. <br/>
Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing. <br/>
-
Sequencing (pIG25 and pIG27) sent.
+
Sequencing (pIG25 and pIG27) sent. <br/><br/>
</p>
</p>
-
 
     <br/> <br/>
     <br/> <br/>
Line 77: Line 102:
<p style=" line-height : 1.5em">
<p style=" line-height : 1.5em">
-
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
+
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/>
 +
Purification and culture of 2 clones from each Petri dish of the transformation of rcn-ompR234 + pSB1C3 in MC4100.<br/>
 +
Extraction of piG33 800µl 1 and 2, and pIG33 200µl 1 and 2.<br/>
 +
Digestion by EcoR1, Pst1 and gel electrophoresis for verification.<br/><br/><br/>
</p>
</p>
Line 84: Line 112:
<p style=" line-height : 1.5em">
<p style=" line-height : 1.5em">
<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/>
<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/>
-
Start of a 5mL liquid culture of S17 in LB+Cm medium<br/>
+
Start of a 5mL liquid culture of S17 in LB+Cm medium.<br/>
-
Minipreps of this culture to extract pIG25 in larger quantity. Digestion (E+P) and electrophoresis for control : the insert is too small, not correct.
+
Minipreps of this culture to extract pIG25 in larger quantity. Digestion (E+P) and electrophoresis for control : the insert of pIG25 is not good so there was a mistake the first time it was controlled. More or less 500bp are missing. Back to the lab, let’s try to built it again to have it on time.<br/>
 +
The inserts pIG33 800µl 1 and 200µl 2 are good. pIG33 800µl 1 becomes pIG33 and is put in the plasmids’ collection.<br/>
 +
MC4100+pIG33 = S33 is stored.
</p>
</p>
-
<p style=" line-height : 1.5em">
 
-
<FONT COLOR="purple"><b> Adherence Test Preparation </b></FONT> <br/><br/>
 
-
 
-
</p>
 
-
 
-
<p style=" line-height : 1.5em">
 
-
<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/>
 
-
 
-
</p>
 
      
      
Line 107: Line 128:
<h6 style="text-align :left"> Wednesday </h6> <HR>
<h6 style="text-align :left"> Wednesday </h6> <HR>
-
     <br/>
+
     <br/><br/>
   <p style=" line-height : 1.5em">
   <p style=" line-height : 1.5em">
-
<FONT COLOR="red"> <b>Transformations and controls </b> </FONT> <br/><br/>
+
<FONT COLOR="red"> <b>Strain construction </b> </FONT> <br/><br/>
The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.<br/><br/>
The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.<br/><br/>
Digestion by E+P of this plasmid and ligation into pSB1C3 overnight<br/><br/>
Digestion by E+P of this plasmid and ligation into pSB1C3 overnight<br/><br/>
 +
Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3 (pIG25) <br/>
Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails. <br/><br/>
Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails. <br/><br/>
 +
Digestion of pIG20 with E and P.<br/>
 +
Gel electrophoresis for control of the insert → good<br/><br/>
 +
Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3.<br/><br/><br/>
</p>
</p>
-
<p style=" line-height : 1.5em">
+
<div style="border:1px solid black;float:left;margin-left:3%; width : 180px">
-
<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/>
+
    <img src="https://static.igem.org/mediawiki/2011/c/c2/2011-03-17_10-23-03.jpg" width="180px"/>  
-
 
+
</div>
-
</p>
+
<p style=" line-height : 1.5em;margin-left:30%">
-
 
+
<FONT COLOR="purple"><b> Adherence Test - pRcn-OmpR234 Characterization  </b></FONT> <br/><br/>
-
<p style=" line-height : 1.5em">
+
-
<FONT COLOR="purple"><b> rcn-OmpR234 adherence test </b></FONT> <br/><br/>
+
Start of a rcn-OmpR234 24 well plate with :<br/>
Start of a rcn-OmpR234 24 well plate with :<br/>
-
S27 ( rcn in pSB1C3 ) without cobalt and with cobalt (25µM) <br/>
+
S27 ( rcn in pSB1C3 ) without cobalt and with cobalt (25µM). <br/>
-
S33 ( rcn-OmpR234 in pSB1C3 ) with increasing concentration of cobalt (0, 10, 25 and 100 µM)
+
S33 ( rcn-OmpR234 in pSB1C3 ) with increasing concentration of cobalt (0, 10, 25 and 100 µM).<br/><br/><br/>
Line 136: Line 159:
<p style=" line-height : 1.5em">
<p style=" line-height : 1.5em">
<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/>
<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/>
-
Plating of PHL644, PHL1256, PHL628 to be sent to other research teams, on request.<br/><br/>
+
Plating of PHL644, PHL1256, PHL628 to be sent to other research teams, on request.
</p>
</p>
Line 149: Line 172:
  <p style=" line-height : 1.5em">
  <p style=" line-height : 1.5em">
 +
 +
<FONT COLOR="red"> <b>Strain construction </b> </FONT> <br/><br/>
 +
None of the transformation of the day before has grown. Let’s try it again.<br/><br/><br/>
 +
 +
</p>
 +
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="purple"><b> Fluorescence Test - rcn Characterization </b></FONT> <br/><br/>
 +
Characterization of rcn-gfp with the kinetic studie.<br/>
 +
3 people are repeating the same 4 erlen of culture with the M63G medium:<br/>
 +
- NM522/p157 + spc + 25µM Co<br/>
 +
- NM522/p157 + spc<br/>
 +
- NM522/p115 + spc + 25µM Co<br/>
 +
- NM522/p115 + spc<br/>
 +
 +
1 ml of spc (spectinomycine) is added<br/>
 +
The culture is sed by 0,5mL of a overnight preculture.<br/><br/>
 +
     </p>   
     </p>   
 +
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="purple"><b> Adherence Test - 18A-0mpR234 Characterization</b></FONT> <br/><br/>
 +
 +
Start a 24 well plates in M63G medium + Amp with PHL1414/piG3 (negative control) and PHL1414/piG16.<br/>
 +
Incubation at 30°C for 48h.<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">
 +
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/>
 +
The transformations from Wednesday have grown : selection of 5 clones for pIG36 and Ptrc +Cm, 10 for pIG25.<br/><br/>
-
    </p>   
+
Miniprep (QIAGen), digestion (Fermentas E+P) and electrophoresis of all clones for pIG25, 4 for pIG36, 2 for Ptrc-Cm ( the other cultures had not grown ). One correct clone is found for pIG25 and pIG36, none is correct for Ptrc-Cm.<br/><br/>
 +
<div style="border:1px solid black;float:left;margin-left:25%; width : 300px">
 +
    <img src="https://static.igem.org/mediawiki/2011/1/19/Gelwiki13.JPG" width="300px"/>   
 +
</div>
 +
</p>
 +
 
 +
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/>
 +
 
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="purple"> <b> Adherence Test - pRcn-OmpR234 Characterization </b> </FONT> <br/><br/>
 +
Revealing of the plate : no effect from rcn-ompR234 is visible compared to the controls. <br/><br/>
 +
 
 +
</p>
 +
 
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/>
 +
Dissolution of 0.11g of Ampiciline sodium salt ( Sigma A0166-5G) into 10mL water and filtration to obtain a solution of Amp at 10mg/mL.
 +
</p>
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
 +
<h6 style="text-align :left"> Saturday </h6><HR><br/><br/>
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/>
 +
Storage of the correct clones and extracted plasmids from the previous day : S34 (NM522 + pIG25), S35 (NM522+pIG36), pIG25, pIG37 (=pIG36 after transformation and extraction ).<br/><br/>
 +
</p> 
 +
 +
<p style=" line-height : 1.5em">
 +
<FONT COLOR="purple"> <b> Adherence Test - 18A-OmpR234 Characterization  </b> </FONT> <br/><br/>
 +
Revealing of the plate : ompR234 doubles the adherence compared to negative controls.<br/><br/>
 +
 +
</p>
 +
 +
    <br/> <br/>
 +
    <br/> <br/>
 +
 +
<h6 style="text-align :left"> Sunday </h6><HR>
 +
 +
<br/><br/>
 +
  <p style=" line-height : 1.5em">
 +
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/>
 +
Miniprep (QIAGen), digestion (Fermentas E+P) and electrophoresis of the last 3 clones for Ptrc-Cm.<br/><br/>
 +
</p>
 +
 +
    <br/> <br/>
 +
    <br/> <br/>
 +
 +
    <br/> <br/>
 +
    <p>
 +
              <a  href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week12"/><font color="grey"><b>Previous Week</b></font></a>
 +
              <a  style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week17"/><font color="grey"><b>Next Week</b></font></a>
 +
              <br/>
 +
    </p>
 +
    <br/> <br/>
</div>
</div>

Latest revision as of 18:50, 27 October 2011







Week 13


From Monday the 12th of September to Sunday the 18th of September 2011







Monday



Strain construction

Extraction of PHL1414+pIG3, PHL1414+pIG16 (clone 1,2,3) , MC4100+piG34 200µl (clone 1,2), MC4100+pIG34 800µl (clone 1, 2).

Digestion by EcoRI and PstI.

Gel electrophoresis to verify the ligations. Inserts have the good length for pIG3, pIG16 so the transformation went well. Inserts of pIG34 200µl clone 1 and pIG34 800µl clone 1 are good so the ligation went well. We’re keeping in collection the pIG34 200µl clone 1 as pIG34 in the plasmids’ collection and MC400+pIG34 as S32 in the strains’ collection.

Cloning and transformation of rcn-ompR234 + pSB1C3 in MC4100.


Plasmid and strain Collection

Plating of S17 from the collection to extract a bigger quantity of pIG25 (pSB1C3 containing rcn-csgBAEFG).

Storage of the correct clone of:
PHL1414 + pIG3 (S30)
PHL1414+pIG16 (S31)
MC4100 + pIG34 (S32)

Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep.


Microscopy Test

Preparation
Sterilization of microscope cover slips in ethanol during a few minutes, then rinsed in sterilized water.

Test
Preparation of plates for the following strains (one repetition):
-PHL1414/piG3 AmpR = negative control without Co and with Co 25µM
-PHL1414/piG16 AmpR without Co, Co 10µM, Co 25µM, Co 50µM and Co 100µM

Incubation at 30°C overnight (during 16 hours).


Others

Pouring of 9 LB+Amp plates

Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing.
Sequencing (pIG25 and pIG27) sent.



Tuesday



Strain construction

Purification and culture of 2 clones from each Petri dish of the transformation of rcn-ompR234 + pSB1C3 in MC4100.
Extraction of piG33 800µl 1 and 2, and pIG33 200µl 1 and 2.
Digestion by EcoR1, Pst1 and gel electrophoresis for verification.


Plasmid and strain Collection

Start of a 5mL liquid culture of S17 in LB+Cm medium.
Minipreps of this culture to extract pIG25 in larger quantity. Digestion (E+P) and electrophoresis for control : the insert of pIG25 is not good so there was a mistake the first time it was controlled. More or less 500bp are missing. Back to the lab, let’s try to built it again to have it on time.
The inserts pIG33 800µl 1 and 200µl 2 are good. pIG33 800µl 1 becomes pIG33 and is put in the plasmids’ collection.
MC4100+pIG33 = S33 is stored.





Wednesday



Strain construction

The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.

Digestion by E+P of this plasmid and ligation into pSB1C3 overnight

Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3 (pIG25)
Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails.

Digestion of pIG20 with E and P.
Gel electrophoresis for control of the insert → good

Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3.


Adherence Test - pRcn-OmpR234 Characterization

Start of a rcn-OmpR234 24 well plate with :
S27 ( rcn in pSB1C3 ) without cobalt and with cobalt (25µM).
S33 ( rcn-OmpR234 in pSB1C3 ) with increasing concentration of cobalt (0, 10, 25 and 100 µM).


Others

Plating of PHL644, PHL1256, PHL628 to be sent to other research teams, on request.





Thursday


Strain construction

None of the transformation of the day before has grown. Let’s try it again.


Fluorescence Test - rcn Characterization

Characterization of rcn-gfp with the kinetic studie.
3 people are repeating the same 4 erlen of culture with the M63G medium:
- NM522/p157 + spc + 25µM Co
- NM522/p157 + spc
- NM522/p115 + spc + 25µM Co
- NM522/p115 + spc
1 ml of spc (spectinomycine) is added
The culture is sed by 0,5mL of a overnight preculture.

Adherence Test - 18A-0mpR234 Characterization

Start a 24 well plates in M63G medium + Amp with PHL1414/piG3 (negative control) and PHL1414/piG16.
Incubation at 30°C for 48h.



Friday



Strain construction

The transformations from Wednesday have grown : selection of 5 clones for pIG36 and Ptrc +Cm, 10 for pIG25.

Miniprep (QIAGen), digestion (Fermentas E+P) and electrophoresis of all clones for pIG25, 4 for pIG36, 2 for Ptrc-Cm ( the other cultures had not grown ). One correct clone is found for pIG25 and pIG36, none is correct for Ptrc-Cm.
















Adherence Test - pRcn-OmpR234 Characterization

Revealing of the plate : no effect from rcn-ompR234 is visible compared to the controls.

Others

Dissolution of 0.11g of Ampiciline sodium salt ( Sigma A0166-5G) into 10mL water and filtration to obtain a solution of Amp at 10mg/mL.





Saturday



Plasmid and strain Collection

Storage of the correct clones and extracted plasmids from the previous day : S34 (NM522 + pIG25), S35 (NM522+pIG36), pIG25, pIG37 (=pIG36 after transformation and extraction ).

Adherence Test - 18A-OmpR234 Characterization

Revealing of the plate : ompR234 doubles the adherence compared to negative controls.





Sunday



Strain construction

Miniprep (QIAGen), digestion (Fermentas E+P) and electrophoresis of the last 3 clones for Ptrc-Cm.







Previous Week Next Week






ENS assystem Biomérieux INSA INSA