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Team:Lyon-INSA-ENS/Realisation/Week7
From 2011.igem.org
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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/> | + | <b>Miniprep</b> of some other parts from the transformed bacteria. <b>Digestion</b> and <b>electrophoresis</b> to control the presence of the right insert.<br/> <br/> |
| - | Miniprep of bacteria with mutated part CsgAB. Digestion to check restriction profile (removal of intern PstI site). | + | <b>Miniprep</b> of bacteria with mutated part CsgAB. <b>Digestion</b> to check restriction profile (removal of intern PstI site). |
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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/> | 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/> | ||
| - | 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. | + | Send of plasmid with the right profile for part CsgAB to <b>sequencing</b>. <b>Extraction</b> of new MC4100 strain's DNA. <b>PCR</b> with fresh DNA and Taq Phusion. |
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| - | Midiprep and nanodrop of the following ligated or newly obtained plasmids :<br/> | + | <b>Midiprep</b> and <b>nanodrop</b> of the following ligated or newly obtained plasmids :<br/> |
NiCoT : 112,2 ng/µL <br/> | NiCoT : 112,2 ng/µL <br/> | ||
2M-2L-Tet : 109.6 ng/µL <br/> | 2M-2L-Tet : 109.6 ng/µL <br/> | ||
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2M-24E : 92.3 ng/µL <br/><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) | + | <b>Fermentas digestion</b> : 2M-2L-Tet (X+P), 2M-2L-Kan (X+P), rcn-curli(E+P), 2M-24E(X+P), Curli(S+P) |
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| - | PCR did not work. | + | <b>PCR</b> did not work. |
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| - | Standard ligation of :<br/> | + | Standard <b>ligation</b> of :<br/> |
-Prcn into Cn backbone<br/> | -Prcn into Cn backbone<br/> | ||
-rcn-curli into Cn backbone<br/> | -rcn-curli into Cn backbone<br/> | ||
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-Pcurli into 2M-2L-Tet <br/><br/> | -Pcurli into 2M-2L-Tet <br/><br/> | ||
| - | TSS transformation into NM522. | + | <b>TSS transformation</b> into NM522. |
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Revision as of 13:04, 13 September 2011
Week 7
From Monday the 25th of July to Friday the 29th of July 2011
Monday
Miniprep of some other parts from the transformed bacteria. Digestion and electrophoresis to control the presence of the right insert.
Miniprep of bacteria with mutated part CsgAB. Digestion to check restriction profile (removal of intern PstI site).
Tuesday
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.
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.
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)
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.
Wednesday
Midiprep and nanodrop of the following ligated or newly obtained plasmids :
NiCoT : 112,2 ng/µL
2M-2L-Tet : 109.6 ng/µL
2M-2L-Kan : 128 ng/µL
18A-OmpR234 : 59.5 ng/µL
rcn-Curli : 116 ng/µL
2M-24E : 92.3 ng/µL
Fermentas digestion : 2M-2L-Tet (X+P), 2M-2L-Kan (X+P), rcn-curli(E+P), 2M-24E(X+P), Curli(S+P)
PCR did not work.
Thursday
Standard ligation of :
-Prcn into Cn backbone
-rcn-curli into Cn backbone
-Pcurli into 2M-2L-Kan
-Pcurli into 2M-2L-Tet
TSS transformation into NM522.
