Team:Paris Saclay/Notebook/August/27

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
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='''Notebook : August 27'''=
='''Notebook : August 27'''=
-
=='''summary'''==  
+
=='''Lab work'''==
-
*We got colonies after the transformation of E.coli with the ligation mix "PnirB_PSB1C3 and RBS_LacZ_Term" 
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==='''A - Aerobic/Anaerobic regulation system'''===
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promoter fnr(activator)nirB plus  RBS_AmilCP+Term_PSB1C3 
+
-
"Pndh*_PSB1C3 plus  RBS_LacZ+Term"
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-
"Pndh*_PSB1C3 plus  RBS_AmilCP+Term"
+
-
so we tested for correct ligation with PCR on colonies
+
-
* We did not get any colonies for the Gibson assembly so we analyzed by gel electrophoresis the parts used for the Gibson assembly to verify their sizes and concentrations.
+
-
* Digestion DnpI purification of the  the PSB1C3 clean for Gibson and  electrophoresis of  it .
+
-
<br>
+
-
=='''lab work'''==
+
===='''Objective : characterize BBa_K1155000 and BBa_K1155004'''====
-
<br>
+
===='''1 - PCR Colony  of ligation Pndh* or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3'''====
-
*'''A.aero/anaerobic regulation system'''<br>
+
-
**''1.Colony PCR on e.coli with FNR Promoter plus RBS_LacZ+Term_PSB1C3 or RBS_AmilCP+Term_PSB1C3  for 8 colonies of each''
+
-
Xiaojing
+
XiaoJing
-
*A1-8: promoter fnr(activator)nirB plus  RBS_LacZ+Term_PSB1C3
+
{|
-
*B1-8: promoter fnr(activator)nirB plus  RBS_AmilCP+Term_PSB1C3
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| style="border:1px solid black;padding:5px;background-color:#DE;" |
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*C1-8: promoter fnr(repressor) plus  RBS_LacZ+Term_PSB1C3
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Transformation of 08/26/13 works. We will do a Colony PCR.
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*D1-8: promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3
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|}
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*Picking of 32 colonies
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*Preparation  Master mix
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We took a single colony and resuspend in 10µL H2O.For each Biobrick we did 6 PCR Colony.
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**H<sub>2</sub>O : 490µL
+
-
**dNTP : 17.5µL
+
-
**VR primer : 17.5µL
+
-
**VF2 primer : 17.5µL
+
-
**DreamTaq buffer 10x : 87.5µL
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-
**DreamTaq enzyme : 7µL
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-
Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
+
Used quantities :  
-
PCR Program :
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* DNA : 2µL
 +
* Mix  : (it was divided in 6 tubes for each promotor with 23µL of mix in each tube)
 +
** Oligo 44 : 17.5µL
 +
** Oligo 43 : 17.5µL
 +
** Buffer Dream Taq : 87.5µL
 +
** dNTP : 17.5µL
 +
** Dream Taq : 7µL
 +
** H2O : 591µL 
-
[[File:PsPcr808.jpg|400px]]
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PCR Program :  
 +
[[File:PsPCR2708.jpg|400px]]
 +
====2 - Electrophoresis of the colony PCR products : Pndh* or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3====
 +
XiaoJing
 +
{|
 +
| style="width:350px;border:1px solid black;" | [[File:Psgel12708.jpg|500px]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 6µL DNA ladder
 +
* Well 2 to 7 : 10µL of NirB with RBS-LacZ-Term in pSB1C3 +2µL of 6X loading dye
 +
* Well 8 to 13 : 10µL of NirB with RBS-Amil CP-Term in pSB1C3 +2µL of 6X loading dye
 +
* Well 14 to 19 : 10µL of Pndh* with RBS-LacZ-Term in pSB1C3 +2µL of 6X loading dye
 +
* Well 20 to 25 : 10µL of Pndh* with RBS-Amil CP-Term in pSB1C3 +2µL of 6X loading dye
 +
* Gel : 1%
 +
|}
 +
Expected size :
 +
* NirB with RBS-LacZ-Term in pSB1C3 : 3474bp
 +
* NirB with RBS-AmilCP-Term in pSB1C3 : 1029bp
 +
* Pndh* with RBS-LacZ-Term in pSB1C3 : 3380bp
 +
* Pndh* with RBS-AmilCP-Term in pSB1C3 : 935bp
-
=====2 - Gel electrophoresis of the colony PCR products=====
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{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size for NirB with RBS-Amil CP-Term in pSB1C3 in well 12, Pndh* with RBS-LacZ-Term in pSB1C3 in well 14, 15, 18 and 19 and Pndh* with RBS-Amil CP-Term in pSB1C3 in well 20, 22 and 25. Nevertheless, electrophoresis shows that these colonies weren't pure. We will purify them by streaking.
 +
|}
 +
 
 +
====3 - Streak colonies of NirB with RBS-Amil CP-Term in pSB1C3, Pfnr with RBS-LacZ-Term in pSB1C3 and Pfnr with RBS-Amil CP-Term in pSB1C3 to purify them====
 +
 
 +
XiaoJing
 +
 
 +
====4 - Culture of strain MG1655Z1 Δfnr::Km containing plasmid pcp20====
 +
 
 +
XiaoJing
{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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| style="width:350px;border:1px solid black;vertical-align:top;" |
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Purification from 08/26/13 works.  
-
*6µL DNA Ladder
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-
*10µL sample per well
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-
*Gel : 0.8%
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|}
|}
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Expected size : 3583bp
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We made culture on LB medium at 42°C for strain MG1655Z1 Δfnr::Km containing plasmid pcp20 to select the bacteria that eliminate the Km cassette. Therefore, we can use this strain to receive the plasmid IGEM pSB1K3. Like this we will select clone Δfnr::Km by streaking them on plate with ampicilin, kanamycin and chloramphenicol plates.
-
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
+
Principle :
-
===='''1 -Gel electrophoresis of  parts of Gibson assembly.'''====
+
 +
Plasmid pcp20 is thermosensitive plasmid which produces a flipase that is able to eliminate antibiotic gene (in our case is Km) at 30°C.
 +
When we shift back the strain at 42°C, the plasmid can not replicate and the bacteria will loose their plasmid.
 +
As a result, we constructed successfully the mutant strain MG1655Z1::Δfnr without any antibiotic resistance.
 +
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
 +
===='''Objective : obtaining FNR, BphR2 proteins'''====
 +
 +
===='''1 - Electrophoresis of RBS-BphR2 Part I, BphR2 Part II, FNR Part I , FNR Part II, RBS-FNR Part I and pSB1C3'''====
 +
 +
XiaoJing
{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
| style="width:350px;border:1px solid black;vertical-align:top;" |
+
Gibson tranformation of the 08/27/13 didn't work. So we did an electrophoresis to check sizes and concentrations of Gibson parts.
-
*Well 1 : 6µL DNA Ladder
+
|}
-
*Well 2 : 1µL of PSB1C3 Clean
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-
*Well 3 : 3µL of RBS_BphR2 part1+1µl of 6X loading dy
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*Well 4 : 3µL of BphR2 part2+1µl of 6X loading dy
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*Well 5: 3µL of RBS_FNR part1 +1µl of 6X loading dy
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*Well 6: 3µL of FNR part1 +1µl of 6X loading dy
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-
*Well 7 : 3µL of FNR part2 +1µl of 6X loading dy
+
-
*Gel : 1.0%
+
{|
 +
| style="width:350px;border:1px solid black;" | [[File:Psgel22708.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 6µL DNA ladder
 +
* Well 2 : 5µL of pSB1C3+1µL of 6X loading dye
 +
* Well 3 : 5µL of RBS-BphR2 Part I+1µL of 6X loading dye
 +
* Well 4 : 5µL of RBS-FNR Part I+1µL of 6X loading dye
 +
* Well 5 : 5µL of FNR Part I+1µL of 6X loading dye
 +
* Well 6 : 5µL of FNR Part II+1µL of 6X loading dye
 +
* Well 7 : 5µL of BphR2 Part II+1µL of 6X loading dye
 +
* Gel : 1%
|}
|}
-
Now we do a Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson.
+
Expected sizes :
-
{| border="1" align="center"
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* pSB1C3 : 2070 bp
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|-o
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* RBS-BphR2 Part I : 197 bp
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|PSB1C3 clean for Gibson
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* BphR2 Part II : 790 bp
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|17µl
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* RBS-FNR Part I : 615 bp
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|-
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* FNR Part I : 597 bp
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|enzyme Dnp1
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* FNR Part II : 200 bp
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|1µl
+
 
-
|-
+
{|
-
|buffer
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| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
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|2µl
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We obtain fragment at the right size for RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I, FNR Part II but not for pSB1C3. We will do a digestion of pSB1C3 by DnpI to clean it.
-
|-
+
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|total
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|20µl
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|}
|}
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: Incubate at 37°C for 1h30mins.
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===='''2 - Digestion of pSB1C3 by DnpI'''====
 +
 
 +
XiaoJing
 +
 
 +
Used quantities :
 +
* pSB1C3 : 17µL
 +
* Buffer : 2µL
 +
* DnpI : 1µL
 +
 
 +
We keep the digestion for 1h30 at 37°C.
 +
 
 +
===='''3 - Electrophoresis of the digestion of pSB1C3 by DnpI'''====
-
=====2 - Gel purification of the PSB1C3 Clean for Gibson=====
+
XiaoJing
-
''2.1 - Migration of 20µl PSB1C3 Clean for Gibson digested by Dnp1 '
 
{|
{|
-
| style="width:350px;border:1px solid black;" | [[]]
+
| style="width:350px;border:1px solid black;" | [[File:Psgel32708.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
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*Well 1 : 6µL DNA Ladder
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* Well 1 : 6µL DNA ladder
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*Well 2 : 1µL of PSB1C3 Clean dig Dnp1(08/06/2013)
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* Well 3 : 20µL of pSB1C3 digested by DnpI + 4µL of 6X loading dye
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*Well 3 : 20µL of  PSB1C3 Clean dig Dnp1(08/27/2013)
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* Gel : 1%
 +
|}
 +
Expected sizes :
 +
* pSB1C3 : 2070 bp
-
*Gel : 1.0%
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtain fragment at the right size for pSB1C3. We will purify it.  
|}
|}
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{| border="1" align="center"
{| border="1" align="center"
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||<big>Previous week</big>
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|[[Team:Paris Saclay/Notebook/August/26|<big>Previous day</big>]]
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
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|[[Team:Paris Saclay/Notebook/July/2|<big>Next day</big>]]
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|[[Team:Paris Saclay/Notebook/August/28|<big>Next day</big>]]
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{{Team:Paris_Saclay/incl_fin}}
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Latest revision as of 01:20, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 27

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000 and BBa_K1155004

1 - PCR Colony of ligation Pndh* or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3

XiaoJing

Transformation of 08/26/13 works. We will do a Colony PCR.

We took a single colony and resuspend in 10µL H2O.For each Biobrick we did 6 PCR Colony.

Used quantities :

  • DNA : 2µL
  • Mix  : (it was divided in 6 tubes for each promotor with 23µL of mix in each tube)
    • Oligo 44 : 17.5µL
    • Oligo 43 : 17.5µL
    • Buffer Dream Taq : 87.5µL
    • dNTP : 17.5µL
    • Dream Taq : 7µL
    • H2O : 591µL

PCR Program :

PsPCR2708.jpg

2 - Electrophoresis of the colony PCR products : Pndh* or NirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3

XiaoJing

Psgel12708.jpg
  • Well 1 : 6µL DNA ladder
  • Well 2 to 7 : 10µL of NirB with RBS-LacZ-Term in pSB1C3 +2µL of 6X loading dye
  • Well 8 to 13 : 10µL of NirB with RBS-Amil CP-Term in pSB1C3 +2µL of 6X loading dye
  • Well 14 to 19 : 10µL of Pndh* with RBS-LacZ-Term in pSB1C3 +2µL of 6X loading dye
  • Well 20 to 25 : 10µL of Pndh* with RBS-Amil CP-Term in pSB1C3 +2µL of 6X loading dye
  • Gel : 1%

Expected size :

  • NirB with RBS-LacZ-Term in pSB1C3 : 3474bp
  • NirB with RBS-AmilCP-Term in pSB1C3 : 1029bp
  • Pndh* with RBS-LacZ-Term in pSB1C3 : 3380bp
  • Pndh* with RBS-AmilCP-Term in pSB1C3 : 935bp

We obtained fragments at the right size for NirB with RBS-Amil CP-Term in pSB1C3 in well 12, Pndh* with RBS-LacZ-Term in pSB1C3 in well 14, 15, 18 and 19 and Pndh* with RBS-Amil CP-Term in pSB1C3 in well 20, 22 and 25. Nevertheless, electrophoresis shows that these colonies weren't pure. We will purify them by streaking.

3 - Streak colonies of NirB with RBS-Amil CP-Term in pSB1C3, Pfnr with RBS-LacZ-Term in pSB1C3 and Pfnr with RBS-Amil CP-Term in pSB1C3 to purify them

XiaoJing

4 - Culture of strain MG1655Z1 Δfnr::Km containing plasmid pcp20

XiaoJing

Purification from 08/26/13 works.

We made culture on LB medium at 42°C for strain MG1655Z1 Δfnr::Km containing plasmid pcp20 to select the bacteria that eliminate the Km cassette. Therefore, we can use this strain to receive the plasmid IGEM pSB1K3. Like this we will select clone Δfnr::Km by streaking them on plate with ampicilin, kanamycin and chloramphenicol plates.

Principle :

Plasmid pcp20 is thermosensitive plasmid which produces a flipase that is able to eliminate antibiotic gene (in our case is Km) at 30°C. When we shift back the strain at 42°C, the plasmid can not replicate and the bacteria will loose their plasmid. As a result, we constructed successfully the mutant strain MG1655Z1::Δfnr without any antibiotic resistance.


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

Objective : obtaining FNR, BphR2 proteins

1 - Electrophoresis of RBS-BphR2 Part I, BphR2 Part II, FNR Part I , FNR Part II, RBS-FNR Part I and pSB1C3

XiaoJing

Gibson tranformation of the 08/27/13 didn't work. So we did an electrophoresis to check sizes and concentrations of Gibson parts.

Psgel22708.jpg
  • Well 1 : 6µL DNA ladder
  • Well 2 : 5µL of pSB1C3+1µL of 6X loading dye
  • Well 3 : 5µL of RBS-BphR2 Part I+1µL of 6X loading dye
  • Well 4 : 5µL of RBS-FNR Part I+1µL of 6X loading dye
  • Well 5 : 5µL of FNR Part I+1µL of 6X loading dye
  • Well 6 : 5µL of FNR Part II+1µL of 6X loading dye
  • Well 7 : 5µL of BphR2 Part II+1µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • pSB1C3 : 2070 bp
  • RBS-BphR2 Part I : 197 bp
  • BphR2 Part II : 790 bp
  • RBS-FNR Part I : 615 bp
  • FNR Part I : 597 bp
  • FNR Part II : 200 bp

We obtain fragment at the right size for RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I, FNR Part II but not for pSB1C3. We will do a digestion of pSB1C3 by DnpI to clean it.

2 - Digestion of pSB1C3 by DnpI

XiaoJing

Used quantities :

  • pSB1C3 : 17µL
  • Buffer : 2µL
  • DnpI : 1µL

We keep the digestion for 1h30 at 37°C.

3 - Electrophoresis of the digestion of pSB1C3 by DnpI

XiaoJing

Psgel32708.jpg
  • Well 1 : 6µL DNA ladder
  • Well 3 : 20µL of pSB1C3 digested by DnpI + 4µL of 6X loading dye
  • Gel : 1%

Expected sizes :

  • pSB1C3 : 2070 bp

We obtain fragment at the right size for pSB1C3. We will purify it.


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