Team:Lyon-INSA-ENS/Realisation/Week7

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  <a href="https://2011.igem.org/Main_Page" >
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      <img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" />
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        <img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Realisation/Week7Fr">Version Fran&ccedil;aise</a>
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     <h1 style="color: white;"> Week 7 </h1>  
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     <h1 style="color: white;"> Week 7 </h1>    
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     <p style="text-align : center"> <small> From Monday the 18th of July to Friday the 22nd of July 2011 </small> </p>
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     <p style="text-align : center"> <small> From Monday the 25th of July to Friday the 29th of July 2011 </small> </p>
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   <h6 style="text-align :left"> Monday </h6> <HR>  
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   <h6 style="text-align :left"> Monday </h6> <HR>
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Concerned parts : RBS-GFP-LVA (Cn),BBa_J23119 ( 18A, plate 1 )(Amp), Curli (Cn), ompR234 (Cn)
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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      <br/><br/>
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Start of 5mL liquid cultures from a Petri dish culture ( 5mL sterile LB, 50µL antibiotic ,bacteria). Incubation at 37°C
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<b>Miniprep</b> of some other parts from the transformed bacteria. <b>Digestion</b> and <b>electrophoresis</b> to control the presence of the right insert.<br/> <br/><br/>
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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 ) )
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</p>
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Incubation at 37°C overnight.
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<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br>
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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/>
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<b>Miniprep</b> of bacteria with mutated part CsgAB. <b>Digestion</b> to check restriction profile (removal of intern PstI site).
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Incubation at 37°C <br/>
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   <h6 style="text-align :left"> Tuesday </h6> <HR>
   <h6 style="text-align :left"> Tuesday </h6> <HR>
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<FONT COLOR="purple"><b> Adherence Tests</b></FONT> <br><br>
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Start of a compared culture of a strain transformed with 18A ( constitutive promoter ) only and 18A+OmpR234 ( part which is supposed to induce the formation of a biofilm ) in LB medium, 37°C to check the qualitative behaviour of the OmpR234 part.<br/>
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OmpR234 part seems to make the bacteria adherent, the experiment was restarted with a different protocol ( LB diluted by 2 in water, 30°C ) to enhance the adherence.<br/> <br/>
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<div style="border:1px solid black;float:left;margin-left:3%; width : 200px">
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    <img src="https://static.igem.org/mediawiki/2011/7/74/Boite-petri.jpg" width="200px"/>   
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Midiprep to extract DNA from the previous liquid cultures.
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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Start of 5mL liquid cultures for minipreps : 18A-OmpR234, 2M-2L Tet, 2M-2L-Kan, rcn-curli, 2M-24E, NiCoT ( a transporter for cobalt that makes the bacteria import cobalt from the medium) <br/> <br/><br/>
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Nanodrop quantification :
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</p>
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OmpR234 : 59.5 ng/µL
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<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br>
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GFP : 109.5 ng/µL
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18A : 73.7 ng/µL
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Curli : 82.6 ng/µL
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The 260/280 ratio is lower than 2 in all cases.<br/> <br/>  
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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.  
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Send of plasmid with the right profile for part CsgAB to <b>sequencing</b>. <b>Extraction</b> of new MC4100 strain's DNA. <b>PCR</b> with fresh DNA and Taq Phusion.
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Digestion of several biobricks : RBS, GFP, YFP, Curli, OmpR234.
 
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Electrophoresis to control the digestions.<br/> <br/>
 
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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.
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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<b>Midiprep</b> and <b>nanodrop</b> of the following ligated or newly obtained plasmids :<br/>
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NiCoT : 112,2 ng/µL <br/>
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2M-2L-Tet : 109.6 ng/µL <br/>
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2M-2L-Kan : 128 ng/µL <br/>
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18A-OmpR234 : 59.5 ng/µL <br/>
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rcn-Curli : 116 ng/µL <br/>
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2M-24E : 92.3 ng/µL <br/><br/>
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<b>Fermentas digestion</b> : 2M-2L-Tet (X+P), 2M-2L-Kan (X+P), rcn-curli(E+P), 2M-24E(X+P), Curli(S+P)
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</p>
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<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br>
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<b>PCR</b> did not work.
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Standard or 3A ligation of the previously cut biobricks.TSS transformation into NM522.<br/>
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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Standard <b>ligation</b> of :<br/>
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Transformation of NM522 with the mutated plasmid of part csgAB.<br/>
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-Prcn into Cn backbone<br/>
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    </p>
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-rcn-curli into Cn backbone<br/>
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-Pcurli into 2M-2L-Kan <br/>
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-Pcurli into 2M-2L-Tet <br/><br/>
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<b>TSS transformation</b> into NM522.
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<h6 style="text-align :left"> Friday </h6> <HR>
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Start of 5mL liquid cultures of selected individual colonies containing the biobricks.
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Extraction of the plasmids to test the presence of the insert. <br/> <br/>
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Liquid culture of transformed colonies with csgAB for miniprep
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<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br>
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PCR with genomic DNA of MC4100 with Taq DNA Polymerase and Phusion
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  <h6 style="text-align :left"> Friday </h6> <HR>
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    <br/> <br/>
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<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br>
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PCR did not work. New try with different concentrations of primers (half, normal and twice)  with Taq DNA polymerase and Phusion.    </p> 
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              <a  href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week6"/><font color="grey"><b>Previous Week</b></font></a>
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              <a  style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week8"/><font color="grey"><b>Next Week</b></font></a>
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Latest revision as of 23:35, 21 September 2011







Week 7


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







Monday

Strain construction

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


PCR and mutagenesis of rcn, csgBA, csgEFG

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



Tuesday


Adherence Tests

Start of a compared culture of a strain transformed with 18A ( constitutive promoter ) only and 18A+OmpR234 ( part which is supposed to induce the formation of a biofilm ) in LB medium, 37°C to check the qualitative behaviour of the OmpR234 part.
OmpR234 part seems to make the bacteria adherent, the experiment was restarted with a different protocol ( LB diluted by 2 in water, 30°C ) to enhance the adherence.


Strain construction

Start of 5mL liquid cultures for minipreps : 18A-OmpR234, 2M-2L Tet, 2M-2L-Kan, rcn-curli, 2M-24E, NiCoT ( a transporter for cobalt that makes the bacteria import cobalt from the medium)


PCR and mutagenesis of rcn, csgBA, csgEFG

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

Strain construction

Midiprep and nanodrop of the following ligated or newly obtained plasmids :
NiCoT : 112,2 ng/µL
2M-2L-Tet : 109.6 ng/µL
2M-2L-Kan : 128 ng/µL
18A-OmpR234 : 59.5 ng/µL
rcn-Curli : 116 ng/µL
2M-24E : 92.3 ng/µL

Fermentas digestion : 2M-2L-Tet (X+P), 2M-2L-Kan (X+P), rcn-curli(E+P), 2M-24E(X+P), Curli(S+P)


PCR and mutagenesis of rcn, csgBA, csgEFG

PCR did not work.





Thursday

Strain construction

Standard ligation of :
-Prcn into Cn backbone
-rcn-curli into Cn backbone
-Pcurli into 2M-2L-Kan
-Pcurli into 2M-2L-Tet

TSS transformation into NM522.




PCR and mutagenesis of rcn, csgBA, csgEFG

PCR with genomic DNA of MC4100 with Taq DNA Polymerase and Phusion





Friday


PCR and mutagenesis of rcn, csgBA, csgEFG

PCR did not work. New try with different concentrations of primers (half, normal and twice) with Taq DNA polymerase and Phusion.





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ENS assystem Biomérieux INSA INSA
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