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Week 9

From Monday the 15th of August to Friday the 19th of August 2011

Monday

Transformations and controls for future tests

Transformation and plating of NM522 with : pIG3 (3µL), pIG16. (3µL)

Tuesday

Transformations and controls for future tests

Plating of individual or non individual clones from the previous NM522-pIG16 and NM522-pIG3 cultures on LB+amp medium.
Incubation at 37°C overnight.

Adherence Test

Start of 5mL cultures for adherence tests from the collection :
LB : NM522
LB/2 : NM522, S3, S4, S15, S19
M63 : NM522, S15, S3, S19, S4, S18

Wednesday

Transformations and controls for future tests

Start of 3x5mL cultures of NM522 in LB medium from 50µL of a saturated NM522 culture for a later transformation.
Start of 5mL cultures of the individual clones plated on the previous day.

Transformation and plating of NM522 on LB+Amp with : pIG6 (3µL), pIG7 (3µL), pIG24 (2µL), p56 (2µL), p10 (2µL), pIG16+p157 (3µL each), pIG16+p115(3µL each), pIG16+p127(3µL each), pIG16+p116 ( 3µL and 5µL respectively ), pIG25 (1µL).

Transformation and plating of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.

Extraction of pIG16 from S19 with the QIAGen miniprep protocol.

Adherence Test

Start of 24 well plate adherence test in M63G medium with PHL818
( positive control ), NM522( negative control ), S18 + Amp + CoCl2
( in increasing concentration )

Others

Test of antibiotics : start of 5mL cultures ( 5mL LB, 50µL antibiotic, 10µL saturated NM522) of NM522 with antibiotics to test if the antibiotic is still good for Kan, Tet, Cm and Amp
The bacteria with Amp grow : a new solution is made ( 5x1mL mother solutions at 100mg/mL from solid Amp, and 2x10mL solutions at 100µg/mL by dilution of 10µL of mother solution into 10mL sterile water ).

Re-synthetising csg-BAEFG with standard iGEM restriction sites

Launched an overnight PCR using the synthetised rcn-CsgBAEFG part as the template, and primers used during the first try to obtain the csg construct : 3' primer for csgBA and 5' primer for csgEFG

Thursday

Transformations and controls for future tests

The transformation gave too many bacteria : plating of all the previous transformed strains on a new Petri dish in order to isolate individual clones.

Digestion of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.
Digestion of pIG16 by E,X,S,P,E+P,X+S : E,X and S show a linearization of the plasmid, P shows nothing, E+P shows a linearization and X+S extracts the part -> P was inactive.

Adherence Test Preparation

Start of 5mL cultures for 24 well plates :
M63 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm)
LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522
LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522

Re-synthetising csg-BAEFG with standard iGEM restriction sites

Revealing of the PCR : it is positive, but the negative control is positive too. PCR re-done.

Friday

Transformations and controls for future tests

Plating of NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116 (who have a double resistance) on their second resistance ( Kan ).
Extraction (QIAgen) and digestion(Fermentas) of these plasmids by E : only one plasmid was in the strains -> the transformations need to be restarted.

The NM522/pIG24 (Cm) strain grows on Amp plates and not Cm. Moreover, pIG24 is a linear plasmid and should not have transformed : plating of NM522, NM522/pIG24 on LB+Amp and start of 5mL cultures ( LB+Amp ) of the same strains to test Amp efficiency or contamination of NM522 with an AmpR strain.

Adherence and Flocculation Test

Start of 24 well plates :
(1) M63 : PHL818 ( positive control ), NM522 and NM522/pUC18( negative controls ), S18 + Co ( in increasing concentration)-> to characterize rcn-csgBAEFG
(2) M63 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak
(3) LB/2 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak

Start of tubes for flocculation : the same as plates (2) and (3).

Re-synthetising csg-BAEFG with standard iGEM restriction sites

Revealing PCR results, they show a ~2800 bp DNA band, in accordance with the expected size.

Previous Week Next Week

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-{{Lyon-INSA-ENS/menuNotebookW9}}
+<!--{{Lyon-INSA-ENS/menuNotebookW9}}-->
+{{Lyon-INSA-ENS/menuNotebookVertical|Week 9 = actif}}
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+<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>
      <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/Week9Fr">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/Week9Fr">Version Fran&ccedil;aise</a>
      </div>
@@ Line 18: / Line 25: @@
      <br/>
-<div class="cadre" ; style="background-color:green;" >
+<div class="cadre" ; style="background-color:green;margin-left : 8%" >
      <br/>
      <h1 style="color: white;"> Week 9 </h1>
@@ Line 35: / Line 42: @@
   <p style=" line-height : 1.5em">
+<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
-<!-- Digestion with E+X, ligation with Transformation and plating of NM522 with : pIG3 (3µL), pIG16 (3µL)<br/> -->
+<b>Transformation and plating</b> of NM522 with : pIG3 (3µL), pIG16. (3µL)<br/>
@@ Line 48: / Line 56: @@
   <p style=" line-height : 1.5em">
+<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
+Plating of individual or non individual clones from the previous NM522-pIG16 and NM522-pIG3 cultures on LB+amp medium.<br/> Incubation at 37°C overnight.<br/><br/><br/>
+</p>
+<p style=" line-height : 1.5em">
+<FONT COLOR="purple"> <b>Adherence Test</b> </FONT> <br/><br/>
 Start of 5mL cultures for adherence tests from the collection : <br/>
 LB : NM522 <br/>
 LB/2 : NM522, S3, S4, S15, S19 <br/>
-M63 : NM522, S15, S3, S19, S4, S18 <br/><br/>
+M63 : NM522, S15, S3, S19, S4, S18
+</p>
-Plating of individual or non individual clones from the previous NM522-pIG16 and NM522-pIG3 cultures on LB+amp medium. Incubation at 37°C overnight.<br/>
 </p>
@@ Line 66: / Line 81: @@
      <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/>
 Start of 3x5mL cultures of NM522 in LB medium from 50µL of a saturated NM522 culture for a later transformation.<br/>
 Start of 5mL cultures of the individual clones plated on the previous day. <br/><br/>
+<b>Transformation</b> and <b>plating</b> of NM522 on LB+Amp with : pIG6 (3µL), pIG7 (3µL), pIG24 (2µL), p56 (2µL), p10 (2µL), pIG16+p157 (3µL each), pIG16+p115(3µL each), pIG16+p127(3µL each), pIG16+p116 ( 3µL and 5µL respectively ), pIG25 (1µL). <br/><br/>
+<b>Transformation</b> and <b>plating</b> of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.<br/><br/>
+<b>Extraction</b> of pIG16 from S19 with the QIAGen miniprep protocol. <br/><br/><br/>
+</p>
+<div style="border:1px solid black;float:left;margin-left:3%; width : 180px">
+     <img src="https://static.igem.org/mediawiki/2011/d/de/14.06_plaque.jpg" width="180px"/>
+ </div>
+    <p style=" line-height : 1.5em;margin-left:30%;">
+<FONT COLOR="purple"> <b>Adherence Test</b> </FONT> <br/><br/>
+Start of <b>24 well plate</b> adherence test in M63G medium with PHL818<br/>( positive control ), NM522( negative control ), S18 + Amp + CoCl2<br/>( in increasing concentration ) <br/> <br/><br/><br/>
+</p>
+ <p style=" line-height : 1.5em">
+<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/>
 Test of antibiotics : start of 5mL cultures ( 5mL LB, 50µL antibiotic, 10µL saturated NM522) of NM522 with antibiotics to test if the antibiotic is still good for Kan, Tet, Cm and Amp<br/>
-The bacteria with Amp grow : a new solution is made ( 5x1mL mother solutions at 100mg/mL from solid Amp, and 2x10mL solutions at 100µg/mL by dilution of 10µL of mother solution into 10mL sterile water )<br/><br/>
+The bacteria with Amp grow : a new solution is made ( 5x1mL mother solutions at 100mg/mL from solid Amp, and 2x10mL solutions at 100µg/mL by dilution of 10µL of mother solution into 10mL sterile water ).
+</p>
+<br/><br/><br/>
+<p style=" line-height : 1.5em">
+<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br>
+Launched an overnight PCR using the synthetised rcn-CsgBAEFG part as the template, and primers used during the first try to obtain the csg construct : 3' primer for csgBA and 5' primer for csgEFG<br>
+    </p>
-Start of <b>24 well plate</b> adherence test in M63G medium with PHL818 ( positive control ), NM522 ( negative control ), S4 + Amp + CoCl2 ( in increasing concentration ) <br/> <br/>
-<b>Transformation</b> and <b>plating</b> of NM522 on LB+Amp with : pIG6 (3µL), pIG7 (3µL), pIG24 (2µL), p56 (2µL), p10 (2µL), pIG16+p157 (3µL each), pIG16+p115(3µL each), pIG16+p127(3µL each), pIG16+p116 ( 3µL and 5µL respectively ), pIG25 (1µL). <br/><br/>
-<b>Transformation</b> and <b>plating</b> of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.<br/><br/>
-<b>Extraction</b> of pIG16 from S19 with the QIAGen miniprep protocol
      </p>
@@ Line 92: / Line 135: @@
   <p style=" line-height : 1.5em">
+<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
 The transformation gave too many bacteria : plating of all the previous transformed strains on a new Petri dish in order to isolate individual clones.<br/><br/>
+<b>Digestion</b> of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.<br/>
+<b>Digestion</b> of pIG16 by E,X,S,P,E+P,X+S : E,X and S show a linearization of the plasmid, P shows nothing, E+P shows a linearization and X+S extracts the part -> P was inactive. <br/><br/><br/>
+</p>
+<p style=" line-height : 1.5em">
+<FONT COLOR="purple"> <b>Adherence Test Preparation</b> </FONT> <br/><br/>
 Start of 5mL cultures for 24 well plates :<br/>
@@ Line 99: / Line 150: @@
 LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522<br/><br/>
-<b>Digestion</b> of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.<br/>
+<br/><br/>
-<b>Digestion</b> of pIG16 by E,X,S,P,E+P,X+S : E,X and S show a linearization of the plasmid, P shows nothing, E+P shows a linearization and X+S extracts the part -> P was inactive. <br/><br/>
+<p style=" line-height : 1.5em">
+<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br>
+Revealing of the PCR : it is positive, but the negative control is positive too. PCR re-done.<br>
+    </p>
      </p>
@@ Line 107: / Line 165: @@
   <h6 style="text-align :left"> Friday </h6><HR>
+<br/>
   <p style=" line-height : 1.5em">
-<b>Plating</b> of NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116 ( who have a double resistance ) on their second resistance ( Kan ).<br/>
+<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
+<b>Plating</b> of NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116 (who have a double resistance) on their second resistance ( Kan ).<br/>
 <b>Extraction</b> (QIAgen) and <b>digestion</b>(Fermentas) of these plasmids by E : only one plasmid was in the strains -> the transformations need to be restarted.<br/><br/>
+The NM522/pIG24 (Cm) strain grows on Amp plates and not Cm. Moreover, pIG24 is a linear plasmid and should not have transformed : plating of NM522, NM522/pIG24 on LB+Amp and start of 5mL cultures ( LB+Amp ) of the same strains to test Amp efficiency or contamination of NM522 with an AmpR strain.<br/><br/><br/>
+</p>
+<p style=" line-height : 1.5em">
+<FONT COLOR="purple"> <b>Adherence and Flocculation Test</b> </FONT> <br/><br/>
 Start of <b>24 well plates</b> :<br/>
-M63 : PHL818 ( positive control ), NM522 and NM522/pUC18( negative controls ), S18 + Co ( in increasing concentration).<br/>
+(1) M63 : PHL818 ( positive control ), NM522 and NM522/pUC18( negative controls ), S18 + Co ( in increasing concentration)-> to characterize rcn-csgBAEFG <br/>
-M63 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )<br/>
+(2) M63 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak<br/>
-LB/2 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )<br/><br/>
+(3) LB/2 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak<br/>
+<br>
+Start of tubes for flocculation : the same as plates (2) and (3).<br>
+<br/><br/>
+<p style=" line-height : 1.5em">
+<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br>
+Revealing PCR results, they show a ~2800 bp DNA band, in accordance with the expected size.<br>
+    </p>
-The NM522/pIG24 (Cm) strain grows on Amp plates and not Cm. Moreover, pIG24 is a linear plasmid and should not have transformed : plating of NM522, NM522/pIG24 on LB+Amp and start of 5mL cultures ( LB+Amp ) of the same strains to test Amp efficiency or contamination of NM522 with an AmpR strain.
      </p>
@@ Line 123: / Line 203: @@
      <br/> <br/>
+    <br/> <br/>
+    <p>
+               <a  href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week8"/><font color="grey"><b>Previous Week</b></font></a>
+               <a  style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week10"/><font color="grey"><b>Next Week</b></font></a>
+               <br/>
+    </p>
+     <br/> <br/>
 </div>