Team:Lyon-INSA-ENS/Realisation/Week9

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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 Francaise</a>
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     <h1 style="color: white;"> Week 7 </h1>     
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     <h1 style="color: white;"> Week 9 </h1>     
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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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     <p style="text-align : center"> <small> From Monday the 15th of August to Friday the 19th of August 2011 </small> </p>
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Miniprep of some other parts from the transformed bacteria. Digestion and electrophoresis to control the presence of the right insert.<br/> <br/>
+
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
 +
 
 +
<b>Transformation and plating</b> of NM522 with : pIG3 (3µL), pIG16. (3µL)<br/>
 +
 
-
Miniprep of bacteria with mutated part CsgAB. Digestion to check restriction profile (removal of intern PstI site).
 
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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/>  
+
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
-
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/>
+
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/>
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<p style=" line-height : 1.5em; margin-left : 35%">
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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/>
+
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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.
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<p style=" line-height : 1.5em">
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    </p>
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<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
 +
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 +
 
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<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>
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    <img src="https://static.igem.org/mediawiki/2011/d/de/14.06_plaque.jpg" width="180px"/>   
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     <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">
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<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 ).
 +
</p>
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 +
<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> 
 +
 
 +
 
 +
 
 +
 
-
Midiprep and nanodrop of the following ligated or newly obtained plasmids :<br/>
 
-
NiCoT : 112,2 ng/µL <br/>
 
-
2M-2L-Tet : 109.6 ng/µL <br/>
 
-
2M-2L-Kan : 128 ng/µL <br/>
 
-
18A-OmpR234 : 59.5 ng/µL <br/>
 
-
rcn-Curli : 116 ng/µL <br/>
 
-
2M-24E : 92.3 ng/µL <br/><br/>
 
-
Fermentas digestion : 2M-2L-Tet (X+P), 2M-2L-Kan (X+P), rcn-curli(E+P), 2M-24E(X+P), Curli(S+P)
 
-
<br/><br/>
 
-
PCR did not work.
 
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Standard ligation of :<br/>
+
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
-
-Prcn into Cn backbone<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/>
-
-rcn-curli into Cn backbone<br/>
+
 
-
-Pcurli into 2M-2L-Kan <br/>
+
<b>Digestion</b> of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.<br/>
-
-Pcurli into 2M-2L-Tet <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/><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/>
 +
M63 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm)<br/>
 +
LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522<br/>
 +
LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522<br/><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 of the PCR : it is positive, but the negative control is positive too. PCR re-done.<br>
 +
 
 +
    </p>
 +
 
 +
 
-
TSS transformation into NM522.
 
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  <h6 style="text-align :left"> Friday </h6>
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  <h6 style="text-align :left"> Friday </h6><HR>
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<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/>
 +
<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/>
 +
(1) M63 : PHL818 ( positive control ), NM522 and NM522/pUC18( negative controls ), S18 + Co ( in increasing concentration)-> to characterize rcn-csgBAEFG <br/>
 +
(2) M63 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak<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> 
 +
 +
 +
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              <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>
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Latest revision as of 23:35, 21 September 2011







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






ENS assystem Biomérieux INSA INSA
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