Team:Paris Saclay/Notebook/August/29

From 2013.igem.org

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{{Team:Paris_Saclay/incl_debut_generique}}
 
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='''Notebook : August 29'''=
 
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=='''summary'''==
 
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*Digetion of  RBS_AmilCP+Term_PSB1C3 and  RBS_LacZ+Term_PSB1C3 by SpeI and PstIand check the size by Gel electrophoresis
 
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* check the size by Gel electrophoresis for Degestion product of promoter fnr(activator)
 
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* We do PCR clonies on Gibson assembly and ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .
 
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* we got blue color of ligation promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in anaerobic condition over night.
 
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<br>
 
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=='''lab work'''==
 
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<br>
 
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*'''A.aero/anaerobic regulation system'''<br>
 
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:<u>Digestion for PSB3K3</u>
 
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<br>
 
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{| border="1" align="center"
 
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|-o
 
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|DNA
 
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|9µl
 
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|-
 
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|Ecoil I
 
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|2µl
 
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|-
 
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|PST I
 
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|2µl
 
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|-
 
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|H2O
 
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|5µl
 
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|-
 
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|buffer
 
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|2µl
 
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|-
 
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|total
 
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|20µl
 
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|}
 
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:<u>Digestion for RBS_LacZ+Term_10(08/09/13)
 
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B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 </u>
 
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<br>
 
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{| border="1" align="center"
 
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|-o
 
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|DNA
 
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|14µl
 
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|-
 
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|xpe I
 
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|2µl
 
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|-
 
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|PST I
 
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|2µl
 
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|-
 
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|buffer
 
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|2µl
 
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|-
 
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|total
 
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|20µl
 
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|}
 
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:<u>Digestion for RBS_LacZ+Term_11(08/09/13)
 
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B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 </u>
 
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<br>
 
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{| border="1" align="center"
 
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|-o
 
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|DNA
 
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|14µl
 
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|-
 
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|xpe I
 
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|2µl
 
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|-
 
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|PST I
 
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|2µl
 
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|-
 
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|buffer
 
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|2µl
 
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|-
 
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|total
 
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|20µl
 
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|}
 
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:<u>Digestion for RBS_LacZ+Term_15(08/09/13)
 
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B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 </u>
 
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<br>
 
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{| border="1" align="center"
 
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|-o
 
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|DNA
 
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|14µl
 
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|-
 
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|xpe I
 
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|2µl
 
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|-
 
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|PST I
 
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|2µl
 
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|-
 
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|buffer
 
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|2µl
 
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|-
 
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|total
 
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|20µl
 
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|}
 
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:<u>Digestion for RBS_AmilCP+Term_9(02/09/13) </u>
 
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<br>
 
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{| border="1" align="center"
 
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|-o
 
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|DNA
 
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|14µl
 
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|-
 
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|xpe I
 
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|2µl
 
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|-
 
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|PST I
 
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|2µl
 
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|-
 
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|buffer
 
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|2µl
 
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|-
 
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|total
 
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|20µl
 
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|}
 
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:<u>Digestion for RBS_AmilCP+Term_12(02/09/13) </u>
 
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<br>
 
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{| border="1" align="center"
 
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|-o
 
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|DNA
 
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|14µl
 
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|-
 
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|xpe I
 
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|2µl
 
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|-
 
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|PST I
 
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|2µl
 
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|-
 
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|buffer
 
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|2µl
 
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|-
 
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|total
 
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|20µl
 
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|}
 
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:<u>Degestion product quantification</u>
 
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:We used the nano drop to measure the DNA in 260nm and we found its concentration
 
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{| border="1" align="center"
 
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|-
 
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|name
 
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| fnr(repressor)dig EcoilI and speI
 
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| fnr nark dig EcoilI and speI
 
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|-
 
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|conc.
 
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|25.0ng/µl
 
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|13.6ng/µl
 
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|}
 
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<br>
 
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===='''2 -Gel electrophoresis of  Degestion product of promoter fnr(activator).'''====
 
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{|
 
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| style="width:350px;border:1px solid black;" | [[]]
 
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| style="width:350px;border:1px solid black;vertical-align:top;" |
 
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*Well 1 : 6µL DNA Ladder
 
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*Well 2 : 3µL of promoter fnr(activator)nirB clone 7 dig SPE I and PstI
 
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*Well 3 : 3µL of promoter fnr(activator)narG clone 6 dig SPE I and PstI
 
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*Well 4 : 3µL of promoter fnr(activator)nark Digested SPE I and PstI
 
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*Well 5: 3µL of promoter fnr(activator)narB Digested SPE I and PstI
 
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*Well 6: 3µL of promoter fnr(activator)narG Digested SPE I and PstI
 
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*Gel : 1.0%
 
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|}
 
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===='''3 -Gel electrophoresis of  Degestion product of RBS_AmilCP+Term and RBS_LacZ+Term.'''====
 
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{|
 
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| style="width:350px;border:1px solid black;" | [[]]
 
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| style="width:350px;border:1px solid black;vertical-align:top;" |
 
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*Well 1 : 6µL DNA Ladder
 
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*Well 2 : 3µL of RBS_AmilCP+Term_9 digested by xpe I and PstI
 
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*Well 3 : 3µL of RBS_AmilCP+Term_12 digested by xpe I and PstI
 
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*Well 4 : 3µL of RBS_LacZ+Term_10 digested by xpe I and PstI
 
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*Well 5: 3µL of  RBS_LacZ+Term_11 digested by xpe I and PstI
 
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*Well 6: 3µL of RBS_LacZ+Term_15 digested by xpe I and PstI
 
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*Gel : 1.0%
 
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|}
 
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====1 - Clony PCR of Gibson assembly.====
 
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*AA 1-8  Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3
 
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*AB 1-8  Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate
 
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*AC 1-8 Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
 
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*AD 1-8 Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3
 
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*AE 1-8 Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate
 
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*AF 1-8 Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate
 
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*4 Colonies from purple clonies on ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3
 
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Pick up single clone dip in 10µL of H2O in PCR tube .
 
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Prepare PCR MIX for 55 tubes:
 
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* Buffer Dream Taq : 137.5µL
 
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* dNTP : 27.5µL
 
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* Oligo 44 : 27.5µL
 
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* Oligo 43: 27.5µL
 
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* Dream Taq : 11µL
 
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* H2O : 1.144mL
 
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Add 2µL bacteria into 23µL PCR MIX in PCR tube
 
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PCR Program
 
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[[File:PsPCRLacZ2108.jpg|400px]]
 
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{| border="1" align="center"
 
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||<big>Previous week</big>
 
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|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 
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|[[Team:Paris Saclay/Notebook/August/30|<big>Next day</big>]]
 
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|}
 
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{{Team:Paris_Saclay/incl_fin}}
 
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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}

Revision as of 00:50, 22 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 28

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000 and Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions

XiaoJing

Purification of 08/28/13 didn't work. We have blue colonies for Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for nirB with RBS-LacZ-Term in PSB1C3 in anaerobic conditions. We will streak these colonies again.

PsPfnr2908.jpg PsNirB2908.jpg

We streak :

  • Pfnr with RBS-Amil CP-Term in PSB1C3 with O2 at 37°C
  • Pfnr with RBS-Amil CP-Term in PSB1C3 without O2 at 37°C
  • Pfnr with RBS-Amil CP-Term in PSB1C3 with O2 at 30°C
  • Pfnr with RBS-Amil CP-Term in PSB1C3 without O2 at 30°C
  • NirB with RBS-LacZ-Term in PSB1C3 with O2 with Xgal at 37°C
  • NirB with RBS-LacZ-Term in PSB1C3 without O2 with Xgal at 37°C

We also purify Pfnr with RBS-Amil CP-Term in PSB1C3 in liquid culture at 37°C using :

  • Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic conditions :
    • LB : 10 mL
    • Clone : ...
  • Pfnr with RBS-Amil CP-Term in PSB1C3 in anaerobic conditions :
    • LB : 50mL
    • Clone : ...

2 - Digestion of Bba_K1155000, Bba_K1155003, Bba_K1155007 by Xbal/PstI

XiaoJing

We used clone 9 and 12 for Bba_K1155003, clone 10, 11 and 15 for Bba_K1155007

Used quantities :

  • Bba_K1155003, Bba_K1155007
    • DNA : 14µL
    • Buffer FD : 2µL
    • XbaI FD : 2µL
    • PstI FD : 2µL
  • Bba_K1155000 :
    • DNA : 5µL
    • Buffer FD : 2µL
    • SpeI FD : 2µL
    • PstI FD : 2µL
    • H2O : 9µL

We let the digestion 30 minutes at 37°C.

3 - Electrophoresis to check the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of RBS-Amil CP-Term clone 9+1µL of 6X loading dye
  • Well 3 : 5µL of RBS-Amil CP-Term clone 12+1µL of 6X loading dye
  • Well 4 : 5µL of RBS-LacZ-Term clone 10+1µL of 6X loading dye
  • Well 5 : 5µL of RBS-LacZ-Term clone 11+1µL of 6X loading dye
  • Well 6 : 5µL of RBS-LacZ-Term clone 15+1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • RBS-LacZ-Term :
  • RBS-Amil CP-Term :
[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 3 : 5µL of Pfnr+1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • Pfnr :

We obtain fragments at the right size. We will purify them.

4 - Gel purification of the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI

XiaoJing

Protocol : Gel purification

Nanodrop :

  • Pfnr : 26.2ng/µL

5 - Electrophoresis to check the gel purification of the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI

Damir

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 2µL of RBS-LacZ-Term clone 10+1µL of 6X loading dye
  • Well 3 : 2µL of RBS-Amil CP-Term clone 9+1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • RBS-LacZ-Term :
  • RBS-Amil CP-Term :

We obtain fragments at the right size for RBS-LacZ-Term. We will ligate it.

5 - Electrophoresis to check the digestion of Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of NirB clone 7+1µL of 6X loading dye
  • Well 3 : 5µL of NarG clone 6+1µL of 6X loading dye
  • Well 4 : 5µL of Bba_K1155006 already digested by SpeI+1µL of 6X loading dye
  • Well 5 : 5µL of Bba_K1155004 already digested by SpeI+1µL of 6X loading dye
  • Well 5 : 5µL of Bba_K1155005 already digested by SpeI+1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • NarK, NarG, NirB : ...

We obtain fragments at the right size for NarK. We will ligate it


((((((((((((((====Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3====

1 - Digestion of Bba_J04450 by EcoRI/PstI

Anaïs

Used quantities :

  • Buffer FD: 2µL
  • H2O : 5µL
  • DNA : 9µL
  • EcoRI FD : 1µL
  • PstI FD : 1µL

We let the digestion at 37°C during 10 minutes ??????

2 - Electrophoresis to check the digestion of Bba_J04450 by EcoRI/PstI

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 3 : 5µL of PSB3K3+1µL of 6X loading dye
  • Gel : 1.0%

expected sizes :

  • PSB3K3 :

We obtain fragments at the right size. We will ligate it.

3 - Gel purification of the digestion of Bba_J04450 by PstI/SpeI

XiaoJing

Protocol : Gel purification

Nanodrop :

  • Pfnr : 7.2ng/µL)))))))))))))))))))))))


A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FRN and BphR2 proteins

1 - Colony PCR of FNR, RBS-FNR and RBS-BphR2 in DH5α

XiaoJing

Transformation of 08/28/13 works. We will do a Colony PCR.

COLONIES PIQUEES DANS 20µL d'eau pour chaque colonie.

Used quantities :

  • DNA : 2µL
  • Mix : (it was divided in 8 tubes for 8 different colonies for each assembly with 23µL of mix in each tube. We do it twice.)
    • Oligo 43 : 27.5µL
    • Oligo 44 : 27.5µL
    • dNTP : 27.5µL
    • Buffer Dream Taq : 137.5µL
    • Dream Taq : 11µL
    • H2O : 1144µL

PsPCR2908.jpg

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