Team:Lyon-INSA-ENS/Realisation/Week5

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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/Week5Fr">Version Fran&ccedil;aise</a>
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     <h1 style="color: white;"> Week 5 </h1>     
     <h1 style="color: white;"> Week 5 </h1>     
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     <p style="text-align : center"> <small> From Monday the 4th of July to Friday the 8th of July 2011 </small> </p>
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     <p style="text-align : center"> <small> From Monday the 11th of July to Friday the 15th of July 2011 </small> </p>
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   <h6 style="text-align :left"> Monday </h6> <HR>
   <h6 style="text-align :left"> Monday </h6> <HR>
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Visit of Centraco : a french nuclear waste treatment plant  <br/><br/><br/>
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Additional minipreps using the QuickPure protocol of the same 6 parts ( 5 replica each ) <br/>
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Digestion as previously<br/><br/>
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Ligation to obtain : RBS ( strong and weak ) + GFP, RBS ( strong and weak ) + YFP. This corresponds respectively to 2M+2L, 5L+2L, 2M+24E, 5L+24E assemblies.<br/><br/>
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</p>
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<p style=" line-height : 1.5em"><FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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Start of a 5mL culture of NM522 cells.<br/>
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Start of 5mL cultures of 18A (Amp), 24E (Amp) and Curli(Cn). Incubation at 37°C <br/>
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Start of 50 mL cultures ( 50mL sterile LB, 500µL antibiotic, 100µL from the 5mL cultures ). Incubation at 37°C overnight.
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   <h6 style="text-align :left"> Tuesday </h6> <HR>
   <h6 style="text-align :left"> Tuesday </h6> <HR>
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Start of a 50mL culture of NM522 from the overnight preculture ( 50mL sterile LB, 250µL of preculture ).<br/>
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Transformation of 5µL ( S series ) or 10 µL ( D series ) from the previous ligations into NM522 (V=15mL) using a CaCl2 chemical transformation. Positive control was done with 1µL Puc18, negative with 5µL water. <br/> <br/>
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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Sequencing results obtained. Mutagenesis of the plasmids with the least errors by quick change method to remove unwanted restriction sites (EcoRI or PstI).<br/>
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<b>Miniprep</b> of the ligations from the previous week to check the insert : 5L+2L, 5L+24E, 2M+2L, 2M+24E, 2L-Tet, 24E-Tet<br/>
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4 clones are chosen for each ligation. <br/>
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<b>Digestion</b> and <b>electrophoresis</b> : no clone seemed to contain the insert. Decision to verify the digestion, ligation and transformation protocols. <br/><br/>
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<b>Midiprep</b> of 18A, 24E and curli parts.<br/>
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Nanodrop analysis : <br/>
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-18A : 16.7 ng/µL <br/>
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-Curli : 40.5 ng/µL <br/>
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-24E : 190.8 ng/µL <br/><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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The sequencing results were not enough good due to too high concentration of mutated plasmid. Resent of dillute sample.<br/> <br/>
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<h6 style="text-align :left"> Wednesday </h6> <HR>
<h6 style="text-align :left"> Wednesday </h6> <HR>
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Another french newspaper <B>"Le progrès"</B> interviewed us.<br><br><br/>
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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Start of 5mL cultures of 22M, 2M, 2L, 5L, OmpR234 and RBS-GFP-LVA ( synthesized part with an unstable GFP )<br/><br/>
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Selection of individual transformed colonies and start of solid and liquid culture.<br/><br/>
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Digestion of PCR/mutagenesis product with DpnI to eliminate parental plasmid. Transformation of NM522 strains with plasmid. Culture and selection on ampicilline plates.
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Start of 50mL cultures for the same parts.<br/><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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We shoot our "Cobalt Buster's Clip" which introduces, in a funny way, our team : students, instructors and advisors.  We dedicate the all afternoon to this shooting.
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<b>Miniprep</b> of the clones with the mutated plasmid to get more plasmid for the next step. <br/><br/>
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<h6 style="text-align :left"> Thursday </h6> <HR>
<h6 style="text-align :left"> Thursday </h6> <HR>
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Miniprep using the QuickPure protocol of the previous liquid cultures.<br/><br/>
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Digestion of the plasmids by X+P.<br/><br/>
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French national day but... we worked.<br/><br/><br/>
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Electrophoresis of the digested and non digested plasmids : <br/>
 
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2M+2L S1 : no DNA <br/>
 
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5L+2L S1 : 800 bp insert <br/>
 
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2M+2L S2 : no DNA <br/>
 
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2M+2L D1 : 800 bp insert <br/>
 
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5L+24E S2 : no insert <br/>
 
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5L+2L D2 : 800 bp insert <br/>
 
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2M+24E S2 : no insert <br/>
 
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2M+24E D2 : 800 bp insert <br/><br/>
 
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However, due to the small size of the RBS, a flaw in our protocol ( choice of antibiotic resistances ) doesn't allow us to detect a difference between 24E or 2L plasmids compared to their ligated equivalents. Thus we can't conclude that the 800 bp inserts correspond to the ligated parts.<br/><br/>
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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Selection of clones on plate and liquid culture for miniprep. Digestion of purified PCR product in Tango buffer to have a better efficiency. Transformation of NM522 with purified plasmids.
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<b>Midiprep</b> of 22M, 2M, 2L, 5L, OmpR234, RBS-GFP-LVA.
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<h6 style="text-align :left"> Friday </h6> <HR>
 
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Miniprep of clones with mutated plasmids. Restriction to check the removal of unwanted restriction sites (EcoRI or PstI). Send of plasmids with the expected profile to sequencing.
 
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              <a  href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week4"/><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/Week6"/><font color="grey"><b>Next Week</b></font></a>
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Latest revision as of 23:35, 21 September 2011







Week 5


From Monday the 11th of July to Friday the 15th of July 2011







Monday

Visit of Centraco : a french nuclear waste treatment plant


Strain construction

Start of 5mL cultures of 18A (Amp), 24E (Amp) and Curli(Cn). Incubation at 37°C
Start of 50 mL cultures ( 50mL sterile LB, 500µL antibiotic, 100µL from the 5mL cultures ). Incubation at 37°C overnight.





Tuesday



Strain construction

Miniprep of the ligations from the previous week to check the insert : 5L+2L, 5L+24E, 2M+2L, 2M+24E, 2L-Tet, 24E-Tet
4 clones are chosen for each ligation.
Digestion and electrophoresis : no clone seemed to contain the insert. Decision to verify the digestion, ligation and transformation protocols.


Midiprep of 18A, 24E and curli parts.
Nanodrop analysis :
-18A : 16.7 ng/µL
-Curli : 40.5 ng/µL
-24E : 190.8 ng/µL


PCR and mutagenesis of rcn, csgBA, csgEFG

The sequencing results were not enough good due to too high concentration of mutated plasmid. Resent of dillute sample.





Wednesday

Another french newspaper "Le progrès" interviewed us.


Strain construction

Start of 5mL cultures of 22M, 2M, 2L, 5L, OmpR234 and RBS-GFP-LVA ( synthesized part with an unstable GFP )

Start of 50mL cultures for the same parts.


PCR and mutagenesis of rcn, csgBA, csgEFG

Miniprep of the clones with the mutated plasmid to get more plasmid for the next step.





Thursday

French national day but... we worked.


Strain construction

Midiprep of 22M, 2M, 2L, 5L, OmpR234, RBS-GFP-LVA.







Previous Week Next Week






ENS assystem Biomérieux INSA INSA
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