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Team:Lyon-INSA-ENS/Realisation/Week5
From 2011.igem.org
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
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| + | Visit of Centraco : a french nuclear waste treatment plant <br/><br/> | ||
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| + | <p style=" line-height : 1.5em"><FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br> | ||
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Start of 5mL cultures of 18A (Amp), 24E (Amp) and Curli(Cn). Incubation at 37°C <br/> | Start of 5mL cultures of 18A (Amp), 24E (Amp) and Curli(Cn). Incubation at 37°C <br/> | ||
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Start of 50 mL cultures ( 50mL sterile LB, 500µL antibiotic, 100µL from the 5mL cultures ). Incubation at 37°C overnight. | Start of 50 mL cultures ( 50mL sterile LB, 500µL antibiotic, 100µL from the 5mL cultures ). Incubation at 37°C overnight. | ||
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<p style=" line-height : 1.5em; margin-left : 30%; length: 300px"> | <p style=" line-height : 1.5em; margin-left : 30%; length: 300px"> | ||
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| + | <FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br> | ||
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<b>Miniprep</b> of the ligations from the previous week to check the insert : 5L+2L, 5L+24E, 2M+2L, 2M+24E, 2L-Tet, 24E-Tet<br/> | <b>Miniprep</b> of the ligations from the previous week to check the insert : 5L+2L, 5L+24E, 2M+2L, 2M+24E, 2L-Tet, 24E-Tet<br/> | ||
4 clones are chosen for each ligation. <br/> | 4 clones are chosen for each ligation. <br/> | ||
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
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| + | <FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br> | ||
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<b>Midiprep</b> of 18A, 24E and curli parts.<br/> | <b>Midiprep</b> of 18A, 24E and curli parts.<br/> | ||
Nanodrop analysis : <br/> | Nanodrop analysis : <br/> | ||
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Start of 50mL cultures for the same parts.<br/><br/> | Start of 50mL cultures for the same parts.<br/><br/> | ||
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| + | </p> | ||
| + | <p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br> | ||
<b>Miniprep</b> of the clones with the mutated plasmid to get more plasmid for the next step. <br/><br/> | <b>Miniprep</b> of the clones with the mutated plasmid to get more plasmid for the next step. <br/><br/> | ||
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<p style=" line-height : 1.5em"> | <p style=" line-height : 1.5em"> | ||
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French national day but... we worked.<br/><br/> | French national day but... we worked.<br/><br/> | ||
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| + | <FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br> | ||
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| + | </p> | ||
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| + | <p style=" line-height : 1.5em"> | ||
<b>Midiprep</b> of 22M, 2M, 2L, 5L, OmpR234, RBS-GFP-LVA. | <b>Midiprep</b> of 22M, 2M, 2L, 5L, OmpR234, RBS-GFP-LVA. | ||
</p> | </p> | ||
Revision as of 15:17, 13 September 2011
Week 5
From Monday the 11th of July to Friday the 15th of July 2011
Monday
Visit of Centraco : a french nuclear waste treatment plant
Strain construction
Start of 5mL cultures of 18A (Amp), 24E (Amp) and Curli(Cn). Incubation at 37°C
Start of 50 mL cultures ( 50mL sterile LB, 500µL antibiotic, 100µL from the 5mL cultures ). Incubation at 37°C overnight.
Tuesday
Strain construction
Miniprep of the ligations from the previous week to check the insert : 5L+2L, 5L+24E, 2M+2L, 2M+24E, 2L-Tet, 24E-Tet
4 clones are chosen for each ligation.
Digestion and electrophoresis : no clone seemed to contain the insert. Decision to verify the digestion, ligation and transformation protocols.
Strain construction
Midiprep of 18A, 24E and curli parts.
Nanodrop analysis :
-18A : 16.7 ng/µL
-Curli : 40.5 ng/µL
-24E : 190.8 ng/µL
The sequencing results were not enough good due to too high concentration of mutated plasmid. Resent of dillute sample.
Wednesday
Start of 5mL cultures of 22M, 2M, 2L, 5L, OmpR234 and RBS-GFP-LVA ( synthesized part with an unstable GFP )
Start of 50mL cultures for the same parts.
PCR and mutagenesis of rcn, csgBA, csgEFG
Miniprep of the clones with the mutated plasmid to get more plasmid for the next step.
Another french newspaper "Le progrès" interviewed us.
Thursday
French national day but... we worked.
Strain construction
Midiprep of 22M, 2M, 2L, 5L, OmpR234, RBS-GFP-LVA.
