Team:Paris Saclay/Notebook/August/29

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{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
 +
='''Notebook : August 29'''=
='''Notebook : August 29'''=
-
=='''summary'''==  
+
=='''Lab work'''==
-
*We got clonies on ligation promoter fnr(activator)nirB plus  RBS_LacZ+Term_PSB1C3 
+
==='''A - Aerobic/Anaerobic regulation system'''===
-
promoter fnr(activator)nirB plus  RBS_AmilCP+Term_PSB1C3 
+
-
promoter fnr(repressor) plus  RBS_LacZ+Term_PSB1C3
+
-
promoter fnr(repressor) plus  RBS_AmilCP+Term_PSB1C3t
+
-
so we do Colony PCR for it
+
-
* We didn't get clonies on Gibson so we do a gel electrophoresis of  parts of Gibson assembly to verify the size
+
-
* Digestion Dnp1 purification of the  the PSB1C3 clean for Gibson and  electrophoresis of  it .
+
-
<br>
+
-
=='''lab work'''==
+
===='''Objective : characterize BBa_K1155000 and BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-
<br>
+
===='''1 - Purification of colonies: NirB with RBS-LacZ-Term in pSB1C3, Pfnr with RBS-AmilCP-Term in pSB1C3 by streaking colonies in aerobic or anaerobic conditions'''====
-
*'''A.aero/anaerobic regulation system'''<br>
+
 +
XiaoJing
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
Purification of 08/28/13 didn't work. We have blue colonies for Pndh* with RBS-AmilCP-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.
 +
|}
-
:<u>Digestion for PSB3K3</u>
+
[[File:PsPfnr2908.jpg|500px]]
-
<br>
+
[[File:PsNirB2908.jpg|500px]]
 +
We streak colonies from construction :
 +
* Pndh* with RBS-Amil CP-Term in pSB1C3 with O2 at 37°C
 +
* Pndh* with RBS-Amil CP-Term in pSB1C3 without O2 at 37°C
 +
* Pndh* with RBS-Amil CP-Term in pSB1C3 with O2 at 30°C
 +
* Pndh* 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
-
{| border="1" align="center"
+
We also purify Pndh* with RBS-AmilCP-Term in pSB1C3 in liquid culture at 37°C using :
-
|-o
+
 
-
|DNA
+
* Pndh* with RBS-AmilCP-Term in pSB1C3 in aerobic conditions :
-
|9µl
+
** LB : 10 mL
-
|-
+
** Clone : 1 and 2
-
|Ecoil I
+
 
-
|2µl
+
* Pndh* with RBS-AmilCP-Term in pSB1C3 in anaerobic conditions :
-
|-
+
** LB : 50mL
-
|PST I
+
** Clone : 1 and 2
-
|2µl
+
 
-
|-
+
===='''2 -  PCR Colony of strain DH5α containing plasmid pSB1C3 with Pndh* and RBS_AmilCP-Term'''====
-
|H2O
+
 
-
|5µl
+
XiaoJing
-
|-
+
 
-
|buffer
+
We resuspend our colonies in 20µL of H2O.
-
|2µl
+
 
-
|-
+
Used quantities :
-
|total
+
* DNA : 2µL of resuspend colony
-
|20µl
+
* PCR mix: 23µL
 +
 
 +
PCR mix:
 +
** Oligo 43 : 14µL
 +
** Oligo 44 : 14µL
 +
** dNTP : 14µL
 +
** Buffer Dream Taq : 69µL
 +
** Dream Taq : 5.5µL
 +
** H2O : 577µL
 +
 
 +
[[File:PsPCR2908.jpg|400px]]
 +
 
 +
===='''3 - 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 incubatethe digestion for 30 minutes at 37°C.
 +
 
 +
===='''4 - Electrophoresis to check the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI'''====
 +
 
 +
XiaoJing
 +
 
 +
{|
 +
| style="width:350px;border:1px solid black;" | [[File:Psgel12908.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 6µL DNA Ladder
 +
* Well 2 : 5µL of RBS-AmilCP-Term clone 9 + 1µL of 6X loading dye
 +
* Well 3 : 5µL of RBS-AmilCP-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%
|}
|}
-
:<u>Digestion for RBS_LacZ+Term_10(08/09/13)
+
Expected sizes :  
-
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 </u>
+
* RBS-LacZ-Term : 3500bp
-
<br>
+
* RBS-AmilCP-Term : 824bp
 +
Expected sizes :
 +
* Pndh* :  111bp
-
{| border="1" align="center"
+
{|
-
|-o
+
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
|DNA
+
We obtained fragments at the right size. We will purify them.
-
|14µl
+
-
|-
+
-
|xpe I
+
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
|}
|}
-
:<u>Digestion for RBS_LacZ+Term_11(08/09/13)
 
-
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 </u>
 
-
<br>
 
 +
===='''5 - Gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI'''====
-
{| border="1" align="center"
+
XiaoJing
-
|-o
+
 
-
|DNA
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
-
|14µl
+
 
-
|-
+
Nanodrop :
-
|xpe I
+
* Pndh* : 26.2ng/µL
-
|2µl
+
 
-
|-
+
===='''6 - Electrophoresis to check the gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI'''====
-
|PST I
+
 
-
|2µl
+
Damir
-
|-
+
 
-
|buffer
+
{|
-
|2µl
+
| style="width:350px;border:1px solid black;" | [[File:Psgel32908.jpg]]
-
|-
+
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
|total
+
* Well 1 : 6µL DNA Ladder
-
|20µl
+
* Well 2 : 2µL of RBS-LacZ-Term clone 10 + 1µL of 6X loading dye
 +
* Well 3 : 2µL of RBS-AmilCP-Term clone 9 + 1µL of 6X loading dye
 +
* Gel : 1.0%
|}
|}
-
:<u>Digestion for RBS_LacZ+Term_15(08/09/13)
+
Expected sizes :  
-
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 </u>
+
* RBS-LacZ-Term : 3500bp
-
<br>
+
* RBS-Amil CP-Term : 824bp
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size for RBS-LacZ-Term. We will ligate it.
 +
|}
-
{| border="1" align="center"
+
===='''7 - 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'''====
-
|-o
+
 
-
|DNA
+
XiaoJing
-
|14µl
+
 
-
|-
+
{|
-
|xpe I
+
| style="width:350px;border:1px solid black;" | [[File:Psgel42908.jpg]]
-
|2µl
+
| style="width:350px;border:1px solid black;vertical-align:top;" |
-
|-
+
* Well 1 : 6µL DNA Ladder
-
|PST I
+
* Well 2 : 5µL of PNirB clone 7 + 1µL of 6X loading dye
-
|2µl
+
* 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
-
|buffer
+
* Well 5 : 5µL of BBa_K1155004 already digested by SpeI + 1µL of 6X loading dye
-
|2µl
+
* Well 6 : 5µL of BBa_K1155005 already digested by SpeI + 1µL of 6X loading dye 
-
|-
+
* Gel : 1.0%
-
|total
+
-
|20µl
+
|}
|}
 +
Expected sizes :
 +
* NarK, NarG, NirB : 2000bp
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size for NarK. We will ligate it
 +
|}
-
:<u>Digestion for RBS_AmilCP+Term_9(02/09/13) </u>
+
===='''8 - Purification colony of strain MG1655Z1 Δfnr '''====
-
<br>
+
 +
XiaoJing
-
{| border="1" align="center"
+
{|
-
|-o
+
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
|DNA
+
Culture from 08/28/13 works. We will do a purification of one colonies on plates with kanamycin, ampicilin and chloramphenicol antibiotics.
-
|14µl
+
-
|-
+
-
|xpe I
+
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
|}
|}
-
:<u>Digestion for RBS_AmilCP+Term_12(02/09/13) </u>
 
-
<br>
 
 +
We streaked 4 colonies in each plate but we used the same colony to streak on plates with LB or kanamycin or ampicilin or chloramphenicol antibiotics. We incubate our colonies at 42°C.
 +
 +
===='''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 incubate the digestion at 37°C for 10 minutes.
 +
 +
===='''2 - Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI'''====
 +
 +
XiaoJing
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" | [[File:Psgel52908.jpg]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 6µL DNA Ladder
 +
* Well 3 : 5µL of PSB3K3 + 1µL of 6X loading dye 
 +
* Gel : 1.0%
 +
|}
 +
 +
Expected sizes :
 +
* pSB3K3 : 2750bp
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtained fragments at the right size. We will ligate it.
 +
|}
 +
 +
===='''3 - Gel purification of the digestion of BBa_J04450 by PstI/SpeI'''====
 +
 +
XiaoJing
 +
 +
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
 +
 +
Nanodrop :
 +
* Pndh* : 7.2ng/µL
 +
 +
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
 +
 +
===='''Objective : obtaining FRN and BphR2 proteins'''====
 +
 +
===='''1 -  PCR Colony of FNR, RBS-FNR and RBS-BphR2 in DH5α strain'''====
 +
 +
XiaoJing
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
Transformation of 08/28/13 works. We will do a Colony PCR.
 +
|}
 +
 +
We mix our colonies in 20µL of H2O.
 +
 +
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
 +
 +
[[File:PsPCR2908.jpg|400px]]
{| border="1" align="center"
{| border="1" align="center"
-
|-o
+
|[[Team:Paris Saclay/Notebook/August/28|<big>Previous day</big>]]
-
|DNA
+
 
-
|14µl
+
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
-
|-
+
 
-
|xpe I
+
|[[Team:Paris Saclay/Notebook/August/30|<big>Next day</big>]]
-
|2µl
+
-
|-
+
-
|PST I
+
-
|2µl
+
-
|-
+
-
|buffer
+
-
|2µl
+
-
|-
+
-
|total
+
-
|20µl
+
|}
|}
 +
 +
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 01:22, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 29

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000 and BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Purification of colonies: NirB with RBS-LacZ-Term in pSB1C3, Pfnr with RBS-AmilCP-Term in pSB1C3 by streaking colonies in aerobic or anaerobic conditions

XiaoJing

Purification of 08/28/13 didn't work. We have blue colonies for Pndh* with RBS-AmilCP-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 colonies from construction :

  • Pndh* with RBS-Amil CP-Term in pSB1C3 with O2 at 37°C
  • Pndh* with RBS-Amil CP-Term in pSB1C3 without O2 at 37°C
  • Pndh* with RBS-Amil CP-Term in pSB1C3 with O2 at 30°C
  • Pndh* 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 Pndh* with RBS-AmilCP-Term in pSB1C3 in liquid culture at 37°C using :

  • Pndh* with RBS-AmilCP-Term in pSB1C3 in aerobic conditions :
    • LB : 10 mL
    • Clone : 1 and 2
  • Pndh* with RBS-AmilCP-Term in pSB1C3 in anaerobic conditions :
    • LB : 50mL
    • Clone : 1 and 2

2 - PCR Colony of strain DH5α containing plasmid pSB1C3 with Pndh* and RBS_AmilCP-Term

XiaoJing

We resuspend our colonies in 20µL of H2O.

Used quantities :

  • DNA : 2µL of resuspend colony
  • PCR mix: 23µL

PCR mix:

    • Oligo 43 : 14µL
    • Oligo 44 : 14µL
    • dNTP : 14µL
    • Buffer Dream Taq : 69µL
    • Dream Taq : 5.5µL
    • H2O : 577µL

PsPCR2908.jpg

3 - 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 incubatethe digestion for 30 minutes at 37°C.

4 - Electrophoresis to check the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI

XiaoJing

Psgel12908.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of RBS-AmilCP-Term clone 9 + 1µL of 6X loading dye
  • Well 3 : 5µL of RBS-AmilCP-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 : 3500bp
  • RBS-AmilCP-Term : 824bp

Expected sizes :

  • Pndh* : 111bp

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

5 - Gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI

XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • Pndh* : 26.2ng/µL

6 - Electrophoresis to check the gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI

Damir

Psgel32908.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 2µL of RBS-LacZ-Term clone 10 + 1µL of 6X loading dye
  • Well 3 : 2µL of RBS-AmilCP-Term clone 9 + 1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • RBS-LacZ-Term : 3500bp
  • RBS-Amil CP-Term : 824bp

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

7 - 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

Psgel42908.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of PNirB 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 6 : 5µL of BBa_K1155005 already digested by SpeI + 1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • NarK, NarG, NirB : 2000bp

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

8 - Purification colony of strain MG1655Z1 Δfnr

XiaoJing

Culture from 08/28/13 works. We will do a purification of one colonies on plates with kanamycin, ampicilin and chloramphenicol antibiotics.

We streaked 4 colonies in each plate but we used the same colony to streak on plates with LB or kanamycin or ampicilin or chloramphenicol antibiotics. We incubate our colonies at 42°C.

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 incubate the digestion at 37°C for 10 minutes.

2 - Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI

XiaoJing

Psgel52908.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 3 : 5µL of PSB3K3 + 1µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • pSB3K3 : 2750bp

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

3 - Gel purification of the digestion of BBa_J04450 by PstI/SpeI

XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • Pndh* : 7.2ng/µL

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

Objective : obtaining FRN and BphR2 proteins

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

XiaoJing

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

We mix our colonies in 20µL of H2O.

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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