Team:Lyon-INSA-ENS/Realisation/Week7

From 2011.igem.org

(Difference between revisions)
Line 6: Line 6:
<html>
<html>
-
 
<body>
<body>
Line 18: Line 17:
<div class="cadre" ; style="background-color:green;" >
<div class="cadre" ; style="background-color:green;" >
     <br/>
     <br/>
-
     <h1 style="color: white;"> Week 7 </h1>  
+
     <h1 style="color: white;"> Week 7 </h1>    
</div>
</div>
<br/>
<br/>
-
     <p style="text-align : center"> <small> From Monday the 18th of July to Friday the 22nd of July 2011 </small> </p>
+
     <p style="text-align : center"> <small> From Monday the 25th of July to Friday the 29th of July 2011 </small> </p>
     <br/> <br/>
     <br/> <br/>
Line 28: Line 27:
     <br/> <br/>
     <br/> <br/>
-
   <h6 style="text-align :left"> Monday </h6> <HR>  
+
   <h6 style="text-align :left"> Monday </h6> <HR>
-
      <br/>
+
 
 +
  <br/>
 +
 
  <p style=" line-height : 1.5em">
  <p style=" line-height : 1.5em">
 +
Miniprep of some other parts from the transformed bacteria. Digestion and electrophoresis to control the presence of the right insert.<br/> <br/>
-
Concerned parts : RBS-GFP-LVA (Cn),BBa_J23119 ( 18A, plate 1 )(Amp), Curli (Cn), ompR234 (Cn)
+
Miniprep of bacteria with mutated part CsgAB. Digestion to check restriction profile (removal of intern PstI site).
-
      <br/><br/>
+
     </p>  
-
Start of 5mL liquid cultures from a Petri dish culture ( 5mL sterile LB, 50µL antibiotic ,bacteria). Incubation at 37°C
+
-
    <br/><br/>
+
-
Later, start of 150mL liquid cultures from the previous 5mL cultures ( 150 mL sterile LB ,300 µL from the grown 5mL bacterial culture, 1.5 mL antibiotic ( Amp or Cn ) )
+
-
Incubation at 37°C overnight.  
+
-
      <br/><br/><br/>
+
-
10X concentration and plating of 100 µL of transformed bacteria from the previous week : 2M+24E ( amp ), Curli + GFP ( Kan ), Kan + 2L ( Kan ), positive control ( Puc18 plasmid ), negative control ( water ) <br/>
+
-
Incubation at 37°C <br/>
+
-
     </p>
+
-
    <br/> <br/>
 
     <br/> <br/>
     <br/> <br/>
-
   <h6 style="text-align :left"> Tuesday </h6> <HR>
+
   <h6 style="text-align :left"> Tuesday </h6>
-
     <br/>   
+
     <br/> <br/>   
      
      
     <p style=" line-height : 1.5em">
     <p style=" line-height : 1.5em">
-
Midiprep to extract DNA from the previous liquid cultures.
+
Send of plasmid with the right profile for part CsgAB to sequencing. Extraction of new MC4100 strain's DNA. PCR with fresh DNA and Taq Phusion.
-
      <br/>
+
-
Nanodrop quantification :
+
-
      <br/>
+
-
OmpR234 : 59.5 ng/µL
+
-
      <br/>
+
-
GFP : 109.5 ng/µL
+
-
      <br/>
+
-
18A : 73.7 ng/µL
+
-
      <br/>
+
-
Curli : 82.6 ng/µL
+
-
      <br/>
+
-
The 260/280 ratio is lower than 2 in all cases.<br/> <br/>
+
-
 
+
-
A critical error in part CsgAB and a less important error in part CsgEFG were detected by sequencing. Restart mutagenesis on another plasmid with part CsgAB without the fatal error. Restart construction of part csgEFG from the beginning. Another clone of CsgEFG is sent to sequencing .Part RcnR is correct.  
+
     </p>   
     </p>   
Line 72: Line 51:
   
   
-
<h6 style="text-align :left"> Wednesday </h6> <HR>
+
<h6 style="text-align :left"> Wednesday </h6>
     <br/>
     <br/>
     <p style=" line-height : 1.5em">
     <p style=" line-height : 1.5em">
-
Digestion of several biobricks : RBS, GFP, YFP, Curli, OmpR234.
+
PCR did not work.
-
Electrophoresis to control the digestions.<br/> <br/>
+
-
 
+
-
Mutagenesis on the other clone of part CsgAB worked. Digestion with DpnI to eliminate parental plasmids. On  the order hand, PCR with Pfu polymerase on MC4100 to amplify part CsgEFG did not work. Retry with extracted DNA of MC4100.
+
     </p>   
     </p>   
     <br/> <br/>
     <br/> <br/>
-
    <br/> <br/>
+
 
    
    
-
<h6 style="text-align :left"> Thursday </h6> <HR>
+
<h6 style="text-align :left"> Thursday </h6>
     <br/>
     <br/>
  <p style=" line-height : 1.5em">
  <p style=" line-height : 1.5em">
-
Standard or 3A ligation of the previously cut biobricks.TSS transformation into NM522.<br/>
 
-
    </p>
 
-
<p style=" line-height : 1.5em">
+
     </p>
-
Transformation of NM522 with the mutated plasmid of part csgAB.<br/>
+
-
     </p>
+
-
    <br/> <br/>
 
     <br/> <br/>
     <br/> <br/>
-
  <h6 style="text-align :left"> Friday </h6> <HR>
+
  <h6 style="text-align :left"> Friday </h6>
-
    <br/>
+
  <p style=" line-height : 1.5em">
  <p style=" line-height : 1.5em">
-
Start of 5mL liquid cultures of selected individual colonies containing the biobricks.
 
-
Extraction of the plasmids to test the presence of the insert. <br/> <br/>
 
-
Liquid culture of transformed colonies with csgAB for miniprep
+
     </p>
-
     </p>  
+
     <br/> <br/>
     <br/> <br/>

Revision as of 07:51, 28 July 2011










Week 7


From Monday the 25th of July to Friday the 29th of July 2011







Monday

Miniprep of some other parts from the transformed bacteria. Digestion and electrophoresis to control the presence of the right insert.

Miniprep of bacteria with mutated part CsgAB. Digestion to check restriction profile (removal of intern PstI site).



Tuesday


Send of plasmid with the right profile for part CsgAB to sequencing. Extraction of new MC4100 strain's DNA. PCR with fresh DNA and Taq Phusion.





Wednesday

PCR did not work.



Thursday



Friday








ENS assystem Biomérieux INSA INSA
Retrieved from "http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week7"