Team:Lyon-INSA-ENS/Realisation/Week12

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     <h1 style="color: white;"> Week 12 </h1>     
     <h1 style="color: white;"> Week 12 </h1>     
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<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
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<b>Miniprep</b> of three individual clones of MC4100/piG6 and MC4100/piG2 isolated on 09/02 in order to chek the presence of the right plasmid.<br/><br/><br/>
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<b>Miniprep</b> of three individual clones of MC4100/piG6 and MC4100/piG2 isolated on 09/02 in order to chek the presence of the right plasmid.<br/>
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<br/><br/>
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  <p style=" line-height : 1.5em;margin-left:30%">
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
<b>TSS transformation</b> of MC4100 with pIG27 (Cm) or pIG16 (Amp).<br/><br/>
<b>TSS transformation</b> of MC4100 with pIG27 (Cm) or pIG16 (Amp).<br/><br/>
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<FONT COLOR="purple"> <b>CFA Test</b> </FONT> <br/><br/>
<FONT COLOR="purple"> <b>CFA Test</b> </FONT> <br/><br/>
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Liquid culture of strains for the test.<br/><br/><br/>
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<p style=" line-height : 1.5em">
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<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/>
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Culture of S22 in LB with Cm (NM522/pIG33) for conservation.
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<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
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Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.<br/><br/>
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Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.<br/><br/><br/>
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Measuring OD600 and Crystal Violet coloration.<br/><br/><br/>
Measuring OD600 and Crystal Violet coloration.<br/><br/><br/>
</p>
</p>
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<p style=" line-height : 1.5em">
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<FONT COLOR="purple"> <b>CFA Test</b> </FONT> <br/><br/>
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CFA test (4 plates) to check stickiness of strains with PHL1414, MC4100, NM522, S18, S20, S21, S23, S24, S25, S26 (10µL of overnight culture)<br/><br/><br/>
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</p>
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<p style=" line-height : 1.5em">
<p style=" line-height : 1.5em">
<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/>
<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/>
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Pouring of Amp plates.
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Pouring of Amp plates.br/><br/>
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No growth of S22 (Too much antibiotic), new culture with the right dose of antibiotics.
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  <p style=" line-height : 1.5em">
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/>
-
E+P <b>digestion and electrophoresis</b> of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.<br/>
+
Extraction with Qiagen’s kit of:<br/>
-
All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation. <br/><br/><br/>
+
- MC4100 pIG27 clones 1, 2, 3 and 4<br/>
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- MC4100 pIG16 clones 1, 2, 3 and 4<br/>
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- MC4100 pIG33 clones 1, 2 and 3<br/>
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E+P <b>digestion (Fermentas) and electrophoresis</b> of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.<br/>
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All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation. <br/><br/>
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<FONT COLOR="purple"> <b>Result of Flocculation Test</b> </FONT> <br/><br/>
<FONT COLOR="purple"> <b>Result of Flocculation Test</b> </FONT> <br/><br/>
Test started on 09/05.<br/>
Test started on 09/05.<br/>
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Crystal Violet coloration for the three series.<br/><br/>
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Crystal Violet coloration for the three series.<br/><br/><br/>
</p>
</p>
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<p style=" line-height : 1.5em">
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<FONT COLOR="purple"> <b>CFA Test</b> </FONT> <br/><br/>
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No significative results with this test<br/><br/><br/>
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<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/>
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No growth of S22 again. Culture of S22 with and without antibiotics.
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/>
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Construction of the composite part rcn+ompR234 in the chassis MC4100. Because both part are in the plasmid pSB1C3, the ligation 3A is done with the plasmid Kanamycine :<br/>
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pIG27 (ES) + pIG11 (XP) + pSB1K3 (EP)<br/><br/>
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An electrophoresis gel is done to verifie the digestion. Everything is ok.<br/>
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A transformation following the TSS transformation protocol is done and the Petri dishes are incubated overnight.<br/><br/>
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Preparation of tubes for sequencing (30µL at approximately 50-100 ng/µL).<br/>
Preparation of tubes for sequencing (30µL at approximately 50-100 ng/µL).<br/>
The pIG27 miniprep is used as such.<br/>  
The pIG27 miniprep is used as such.<br/>  
-
pIG25 is diluted from 5µL of the collection (371.4 ng/µL) with 25µL water.
+
pIG25 is diluted from 5µL of the collection (371.4 ng/µL) with 25µL water.<br/><br/><br/>
     </p>   
     </p>   
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<p style=" line-height : 1.5em">
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<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/>
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No growth of S22 neither with antibiotic nor without antibiotic. We drop the conservation of the plasmid and restart the construction of pIG33 (fusion between promoter Rcn and ompR234).
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<h6 style="text-align :left"> Saturday </h6><HR>
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<p style=" line-height : 1.5em">
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/>
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Results of the ligation from the 09/09/2011<br/>
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- Petri Dish 800µl E.coli → 40 clones<br/>
 +
- Petri dish 200µl E.coli → 12 clones<br/><br/>
 +
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4 clones of each dish are purified.<br/><br/>
 +
 +
Results of the transformation of pIG3 in PHL1414:<br/>
 +
- Witness - (PHL1414 alone) → nothing<br/>
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- PHL1414 + pIG3 → 1 clone<br/>
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- PHL1414 +pIG16 → a lot of clones<br/><br/>
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Purification of the clone PHL1414+pIG3 and purification of 4 clones of PHL1414+pIG16
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              <a  href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week11"/><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/Week13"/><font color="grey"><b>Next Week</b></font></a>
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Latest revision as of 23:36, 21 September 2011







Week 12


From Monday the 5th of September to Friday the 9th of September 2011







Monday

Transformations and controls for future tests

Miniprep of three individual clones of MC4100/piG6 and MC4100/piG2 isolated on 09/02 in order to chek the presence of the right plasmid.


Results of Flocculation Test and New Test

Crystal Violet coloration for the series of 09/02.

Start of a fourth test in M63G medium with :
-MC4100/piG6 (AmpR) without Co and with Co 10µM (negative controls)
-MC4100/piG2 (AmpR) without Co and with Co 10µM, 25µM, 50µM, 100µM.
(three repetitions)

These liquid cultures are let for two days at 30°C and low stirring (75).


Adherence Test – pRcn-csgBAEFG Characterization

Start of three replica of 24 well plates in M63G medium + Amp with MC4100/piG6 without Co and with Co 10µM
(negative control) and MC4100/piG2 without Co and with Co 10µM, 25µM, 50µM, 100µM.
Incubation at 30°C for 48h.

Revealing of the well plates started on 09/02.
Measuring OD600 and Crystal Violet coloration.




Tuesday


Transformations and controls for future tests

TSS transformation of MC4100 with pIG27 (Cm) or pIG16 (Amp).

Digestion and electrophoresis of the previously extracted DNA of MC4100+pIG6 clones and MC4100+pIG2 clones. Comparison made with the parts put in the collection.--> Verification is OK.


Strain Collection

Storage of the strains :
-S25 = MC4100/piG6 (clone #3)
-S26 = MC4100/piG2 (clone #3)


Microscopy Test

Preparation
Start of 5mL liquid cultures of PHL1414/piG2 (AmpR), PHL1414/piG6 (AmpR) from solid cultures (09/02)
and S19/p127 (KanR/AmpR), S19/p116 (KanR/AmpR) from solid cultures (08/23).

Test
Preparation of plates for the following strains (two repetitions):
-S19/p127 KanR/AmpR
-S19/p116 KanR/AmpR = negative control
(18A-OmpR234 Characterization)
-PHL1414/piG6 AmpR = negative control without Co and with Co 10µM
-PHL1414/piG2 AmpR without Co and with Co 10µM, Co 25µM, Co 50µM, Co 100µM
(pRcn-csgBAEFG Characterization)
Incubation at 30°C overnight (for 15 hours).


CFA Test

Liquid culture of strains for the test.


Others

Culture of S22 in LB with Cm (NM522/pIG33) for conservation.





Wednesday

Transformations and controls for future tests

Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.


Results of Microscopy Test

Observation of the different slides with a fluorescent microscope.
There is a problem with S19/p127 and S19/p116 so we start again the same test for these two strains.


Results of Adherence Test - pRcn-csgBAEFG Characterization

Revealing of the well plates started on Monday.
Measuring OD600 and Crystal Violet coloration.


CFA Test

CFA test (4 plates) to check stickiness of strains with PHL1414, MC4100, NM522, S18, S20, S21, S23, S24, S25, S26 (10µL of overnight culture)


Others

Pouring of Amp plates.br/>
No growth of S22 (Too much antibiotic), new culture with the right dose of antibiotics.





Thursday

Transformations and controls for future tests

Extraction with Qiagen’s kit of:
- MC4100 pIG27 clones 1, 2, 3 and 4
- MC4100 pIG16 clones 1, 2, 3 and 4
- MC4100 pIG33 clones 1, 2 and 3
E+P digestion (Fermentas) and electrophoresis of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.
All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation.




Plasmid Collection

Storage of clone 1 for pIG27 and pIG16.
Plating of those same clones on LB+antibiotic (Cm and Amp respectively ).


Results of Microscopy Test

Observation of the different slides with a fluorescent microscope.


Result of Flocculation Test

Test started on 09/05.
Crystal Violet coloration for the three series.


CFA Test

No significative results with this test


Others

No growth of S22 again. Culture of S22 with and without antibiotics.





Friday

Strain construction

Construction of the composite part rcn+ompR234 in the chassis MC4100. Because both part are in the plasmid pSB1C3, the ligation 3A is done with the plasmid Kanamycine :
pIG27 (ES) + pIG11 (XP) + pSB1K3 (EP)

An electrophoresis gel is done to verifie the digestion. Everything is ok.
A transformation following the TSS transformation protocol is done and the Petri dishes are incubated overnight.

Sequencing

Nanodrop quantification of the previous pIG27 miniprep : 48.0 ng/µL.
Preparation of tubes for sequencing (30µL at approximately 50-100 ng/µL).
The pIG27 miniprep is used as such.
pIG25 is diluted from 5µL of the collection (371.4 ng/µL) with 25µL water.


Others

No growth of S22 neither with antibiotic nor without antibiotic. We drop the conservation of the plasmid and restart the construction of pIG33 (fusion between promoter Rcn and ompR234).





Saturday

Strain construction

Results of the ligation from the 09/09/2011
- Petri Dish 800µl E.coli → 40 clones
- Petri dish 200µl E.coli → 12 clones

4 clones of each dish are purified.

Results of the transformation of pIG3 in PHL1414:
- Witness - (PHL1414 alone) → nothing
- PHL1414 + pIG3 → 1 clone
- PHL1414 +pIG16 → a lot of clones

Purification of the clone PHL1414+pIG3 and purification of 4 clones of PHL1414+pIG16







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