Team:Paris Saclay/Notebook/August/28

From 2013.igem.org

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : August 28'''=
='''Notebook : August 28'''=
-
=='''summary'''==  
+
=='''Lab work'''==
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*We got purple clonies on ligation promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3
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==='''A - Aerobic/Anaerobic regulation system'''===
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so we do a purify of 4 clonies in two plate.incubated at 37°C in aerobic and anaerobic condition over night to verify the different.
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*Prepare LB plates with Chlorenphenicol and Xgal .Pufify single conle of promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 and promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 to see if color will change.
+
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* Gel extration of Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson and do Gibson assembly again .
+
===='''Objective : characterize BBa_K1155000 and BBa_K1155004'''====
-
<br>
+
-
=='''lab work'''==
+
===='''1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in pSB1C3, NirB with RBS-Amil CP-Term in pSB1C3, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions'''====
-
<br>
+
XiaoJing
-
'''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''
+
-
*''1 - Gel extraction of the PSB1C3 Clean for Gibson.''
+
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel extraction|Gel extraction]]
 
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*''2- Migration of 3µl PSB1C3 Clean for Gibson (product gel extraction) '
 
{|
{|
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| style="width:350px;border:1px solid black;" | [[]]
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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Transformation of 08/26/13 works. We will use it to characterize all ligations. PCR colony of 08/27/13 works for . We also will use them to characterize ligations : NirB with RBS-Amil CP-Term in pSB1C3, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3.
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*Well 1 : 6µL DNA Ladder
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*Well 2 : 3µL of PSB1C3 Clean dig Dnp1 gel extraction
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-
 
+
-
 
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*Gel : 1.0%
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|}
|}
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*''3- product quantification.''
 
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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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| PSB1C3 Clean dig Dnp1 gel extraction
 
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|-
 
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|conc.
 
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|37.2ng/µl
 
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|}
 
-
 
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<br>
 
-
 
-
 
-
 
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*''4 - Gibson assembly.''
 
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* RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
 
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* FNR_part1, FNR part1 and plasmid PSB1C3
 
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* RBS_FNR part1, FNR_part2 and plasmid PSB1C3
 
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-
 
-
 
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Sample Volume:
 
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{|
 
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| style="width:700px;border:1px solid black;vertical-align:top;" |
 
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*Tube 1 : RBS_BphR2 part1(1ul), BphR2 part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
 
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*Tube 2 : FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
 
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*Tube 3 : RBS_FNR part1(1ul), FNR part2(1ul) , plasmid PSB1C3(3ul),MIX Gibson (15 ul)
 
-
|}
 
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These 3 mixture is incubated at 50°C for up to one hour.
 
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Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.
 
-
 
-
 
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<br>
 
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'''A - Aerobic/Anaerobic regulation system '''
 
-
 
-
*''1 - Purify single clone of promoter fnr(activator)nirB + RBS_LacZ+Term_PSB1C3  on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in anaerobic condition over night. ''
 
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Protocol : [[Team:Paris_Saclay/Protocols/Purify single clone|Purify single clone]]
 
-
 
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*''2 - Purify single clone of promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night''
 
-
 
-
*''3 - Purify single clone  of promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night''
 
-
 
-
 
-
*''4 - Purify 4 single purple colony  of promoter  fnr(repressor)+ RBS_AmilCP+Term_PSB1C3 on two plates wiht Chlorenphenicol .incubated at 37°C in aerobic and anaerobic condition over night''
 
-
 
-
 
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*''5- Purify single clone  of B5 promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night''
 
-
 
-
 
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*''6- Purify single clone  of D1, D5, D6 promoter  fnr(repressor)+ RBS_AmilCP+Term_PSB1C3 on two plates wiht Chlorenphenicol .incubated at 37°C in aerobic and anaerobic condition over night''
 
-
 
-
 
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*''7- Purify single clone of C1, C2, C5 ,C6promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night''
 
-
<br>
 
-
 
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{| border="1" align="center"
 
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||<big>Previous week</big>
 
-
 
-
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 
-
 
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|[[Team:Paris Saclay/Notebook/August/29|<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}}
 
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-
='''Notebook : August 28'''=
 
-
 
-
=='''Lab work'''==
 
-
 
-
==='''A - Aerobic/Anaerobic regulation system'''===
 
-
===='''Objective : characterize Bba_K1155000 and Bba_K1155004'''====
+
We streak colonies from construction :  
 +
* Pndh* with RBS-LacZ-Term in pSB1C3 with O2 and Xgal
 +
* Pndh* with RBS-Amil CP-Term in pSB1C3 with O2
 +
* Pndh* with RBS-Amil CP-Term in pSB1C3 without O2
 +
* NirB with RBS-LacZ-Term in pSB1C3 without O2 and Xgal
 +
* NirB with RBS-Amil CP-Term in pSB1C3 with O2
 +
* NirB with RBS-Amil CP-Term in pSB1C3 without O2
-
===='''1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions'''====
+
===='''2 - Culture of mutant strain MG1655Z1 Δfnr''' ====
XiaoJing
XiaoJing
Line 115: Line 32:
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DE;" |
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
Transformation of 08/26/13 works. We will use it to characterize all ligations. PCR colony of 08/27/13 works for . We also will use them to characterize ligations : NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3.
+
Transformation of 08/27/13 works. We will do a purification on LB plates.
|}
|}
 +
We streaked our colonies on plates with LB and incubated them at 42°C.
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
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===='''Objective : obtaining FRN and BphR2 proteins'''====
===='''Objective : obtaining FRN and BphR2 proteins'''====
-
===='''1 - Gel purification of PSB1C3 digested by DnpI '''====
+
===='''1 - Gel purification of pSB1C3 digested by DnpI '''====
XiaoJing
XiaoJing
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :  
Nanodrop :  
-
* PSB1C3 : 37.2ng/µL
+
* pSB1C3 : 37.2ng/µL
-
===='''2 - Electrophoresis to check the gel purification of PSB1C3 digested by DnpI'''====
+
===='''2 - Electrophoresis to check the gel purification of pSB1C3 digested by DnpI'''====
XiaoJing  
XiaoJing  
{|
{|
-
| style="width:350px;border:1px solid black;" |]]
+
| style="width:350px;border:1px solid black;" |[[File:Psgel12808.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
* Well 1 : 6µL DNA Ladder
* Well 1 : 6µL DNA Ladder
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* Well 2 : 3µL of PSB1C3 digested by DpnI+1µl of 6X loading dye
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* Well 2 : 3µL of pSB1C3 digested by DpnI + 1µl of 6X loading dye
* Gel : 1%
* Gel : 1%
|}
|}
expected sizes :  
expected sizes :  
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* PSB1C3 : 2070 bp
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* pSB1C3 : 2070 bp
{|
{|
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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We obtain frragment at the right size. The gel purification was good. We will use it for Gibson assembly.
+
We obtained a fragment at the right size. The gel purification was good. We will use it for Gibson assembly.
|}
|}
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* RBS-BphR2 :  
* RBS-BphR2 :  
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** PSB1C3 : 3µL
+
** pSB1C3 : 3µL
** BphR2 Part I : 1µL  
** BphR2 Part I : 1µL  
** BphR2 Part II : 1µL  
** BphR2 Part II : 1µL  
-
** Gibson mix : 15µL ???????????????
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** Gibson mix : 15µL  
* FNR :  
* FNR :  
-
** PSB1C3 : 3µL
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** pSB1C3 : 3µL
** FNR Part I : 1µL
** FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
-
** Gisbon mix : 15µL ???????????????
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** Gisbon mix : 15µL  
* RBS-FNR :
* RBS-FNR :
-
** PSB1C3 : 3µL
+
** pSB1C3 : 3µL
** RBS-FNR Part I : 1µL
** RBS-FNR Part I : 1µL
** FNR Part II : 1µL
** FNR Part II : 1µL
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** Gibson mix : 15µL ????????????
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** Gibson mix : 15µL  
-
We let these mix at 50°C during 1h.  
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We incubate these mix at 50°C during 1h inside PCR machine.
-
===='''2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α'''====
+
===='''2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α strain'''====
XiaoJing  
XiaoJing  
-
Protocol : [[Team:Paris_Saclay/Protocols/Bacterial transformation|Bacterial transformation]]
+
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
 +
 
 +
 
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/27|<big>Previous day</big>]]
 +
 
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 
 +
|[[Team:Paris Saclay/Notebook/August/29|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 01:20, 5 October 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

1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in pSB1C3, NirB with RBS-Amil CP-Term in pSB1C3, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions

XiaoJing

Transformation of 08/26/13 works. We will use it to characterize all ligations. PCR colony of 08/27/13 works for . We also will use them to characterize ligations : NirB with RBS-Amil CP-Term in pSB1C3, Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3.

We streak colonies from construction :

  • Pndh* with RBS-LacZ-Term in pSB1C3 with O2 and Xgal
  • Pndh* with RBS-Amil CP-Term in pSB1C3 with O2
  • Pndh* with RBS-Amil CP-Term in pSB1C3 without O2
  • NirB with RBS-LacZ-Term in pSB1C3 without O2 and Xgal
  • NirB with RBS-Amil CP-Term in pSB1C3 with O2
  • NirB with RBS-Amil CP-Term in pSB1C3 without O2

2 - Culture of mutant strain MG1655Z1 Δfnr

XiaoJing

Transformation of 08/27/13 works. We will do a purification on LB plates.

We streaked our colonies on plates with LB and incubated them at 42°C.

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

Objective : obtaining FRN and BphR2 proteins

1 - Gel purification of pSB1C3 digested by DnpI

XiaoJing

Protocol : Gel purification

Nanodrop :

  • pSB1C3 : 37.2ng/µL

2 - Electrophoresis to check the gel purification of pSB1C3 digested by DnpI

XiaoJing

Psgel12808.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 3µL of pSB1C3 digested by DpnI + 1µl of 6X loading dye
  • Gel : 1%

expected sizes :

  • pSB1C3 : 2070 bp

We obtained a fragment at the right size. The gel purification was good. We will use it for Gibson assembly.

3 - Gibson assembly

XiaoJing

Used quantities :

  • RBS-BphR2 :
    • pSB1C3 : 3µL
    • BphR2 Part I : 1µL
    • BphR2 Part II : 1µL
    • Gibson mix : 15µL
  • FNR :
    • pSB1C3 : 3µL
    • FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gisbon mix : 15µL
  • RBS-FNR :
    • pSB1C3 : 3µL
    • RBS-FNR Part I : 1µL
    • FNR Part II : 1µL
    • Gibson mix : 15µL

We incubate these mix at 50°C during 1h inside PCR machine.

2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α strain

XiaoJing

Protocol : Bacterial transformation


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