Team:Lyon-INSA-ENS/Realisation/Week4

From 2011.igem.org

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     <p style="text-align : center"> <small> From Monday the 27th of June to Friday the 1st of July 2011 </small> </p>
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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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   <h6 style="text-align :left"> Monday </h6> <HR>
   <h6 style="text-align :left"> Monday </h6> <HR>
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Another PCR is launched to collect more DNA (Failure).<br/>
 
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Culture of the wrong strains for the transformation...(Failure : the strain already had a plasmid)<br/>
 
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Alcoholic precipitation of PCR product from the previous week.<br/>
 
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  <h6 style="text-align :left"> Tuesday </h6> <HR>
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    <p style = "line-height : 1.5em">
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  <br/>
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Additional minipreps using the QuickPure protocol of the same 6 parts ( 5 replica each ) <br/>
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<p style = "line-height : 1.5em">
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Digestion as previously<br/><br/>
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Culture of NM522 cells for later transformations and Curli for extraction. <br/><br/>
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Ligation of PCR products in pGem-T easy vector to make them standard parts. Transformation in NM522 strain of the ligation product. Selection on ampicilline, X-Gal, IPTG plate. Only CsgAB was not transformed this day.
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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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Start of a 5mL culture of NM522 cells.<br/>
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  <h6 style="text-align :left"> Tuesday </h6> <HR>
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<h6 style="text-align :left"> Wednesday </h6> <HR>
 
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     <p style = "line-height : 1.5em">
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Miniprep, digestion and electrophoresis of the Curli plasmid.<br/><br/>
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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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Selection of white colonies on the plate for part RcnR and CsgEFG. Liquid culture for miniprep.<br/><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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Transformation in NM522 of part CsgAB.
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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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  </p>
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<h6 style="text-align :left"> Thursday </h6> <HR>
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<h6 style="text-align :left"> Wednesday </h6> <HR>
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      <p style = "line-height : 1.5em">
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CaCl2 chemical transformation of some iGEM kit distribution DNA : <br/><br/>
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<U>Plate 1 : </U><br/>
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    <div style="border:1px solid black;float:right;margin-right:6%;">
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18A (constitutive promoter, Amp )<br/>
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        <div style="border : 3px solid green;">
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2M ( strong RBS, Amp ) <br/>
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          <div style="border : 1px solid black;">
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5L ( weak RBS, Amp )<br/>
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              <img src="https://static.igem.org/mediawiki/2011/d/d3/Notebook-tournage.jpg" width="80px" />
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22M ( RBS+YFP, Amp)<br/><br/>
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<U>Plate 2 : </U><br/>
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24E (YFP, Amp + Kan ) <br/>
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2L (GFP, Amp ) <br/><br/>
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Miniprep of clones with CsgEFG and RcnR. Digestion of plasmid with EcoRI to check part insertions. <br/>
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    <p style = "line-height : 1.5em; width : 560px">
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    </p>
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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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  </p>
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     <p style = "line-height : 1.5em;width : 560px">
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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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The local newspaper "VIVA" interviewed us. They ask about our project, our team and about iGEM's organization.
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  </p>
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    </p>
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     <br/> <br/>
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<h6 style="text-align :left"> Thursday </h6> <HR>
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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">
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Miniprep using the QuickPure protocol of the previous liquid cultures.<br/><br/>
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<p style = "line-height : 1.5em">
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Digestion of the plasmids by X+P.<br/><br/>
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Miniprep using the QuickPure kit of the previous 6 iGEM parts. <br/><br/>
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Digestion with :<br/>
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Electrophoresis of the digested and non digested plasmids : <br/>
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S + P : 18A, 2M, 5L<br/>
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2M+2L S1 : no DNA <br/>
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X + P : 22M, 2L, 24E<br/><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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Electrophoresis of the digested plasmids versus the non digested.<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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All the strains had the correct plasmid : they were plated on LB + amp medium.<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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Miniprep of clones with Csg AB. Digestion of plasmid with EcoRI. Send to GATC for sequencing.
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  </p>
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    <br/> <br/>
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    <br/> <br/>
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  </p>
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<h6 style="text-align :left"> Friday </h6> <HR>
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<p style = "line-height : 1.5em;width : 560px">
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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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</p>
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{{Lyon-INSA-ENS/footer}}
{{Lyon-INSA-ENS/footer}}

Revision as of 07:47, 28 July 2011










Week 4


From Monday the 4th of July to Friday the 8th of July 2011







Monday

Additional minipreps using the QuickPure protocol of the same 6 parts ( 5 replica each )
Digestion as previously

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.

Start of a 5mL culture of NM522 cells.





Tuesday

Start of a 50mL culture of NM522 from the overnight preculture ( 50mL sterile LB, 250µL of preculture ).
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.

Sequencing results obtained. Mutagenesis of the plasmids with the least errors by quick change method to remove unwanted restriction sites (EcoRI or PstI).





Wednesday

Selection of individual transformed colonies and start of solid and liquid culture.

Digestion of PCR/mutagenesis product with DpnI to eliminate parental plasmid. Transformation of NM522 strains with plasmid. Culture and selection on ampicilline plates.


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.





Thursday

Miniprep using the QuickPure protocol of the previous liquid cultures.

Digestion of the plasmids by X+P.

Electrophoresis of the digested and non digested plasmids :
2M+2L S1 : no DNA
5L+2L S1 : 800 bp insert
2M+2L S2 : no DNA
2M+2L D1 : 800 bp insert
5L+24E S2 : no insert
5L+2L D2 : 800 bp insert
2M+24E S2 : no insert
2M+24E D2 : 800 bp insert

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.

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.





Friday

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.








ENS assystem Biomérieux INSA INSA
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