Team:Paris Saclay/Notebook/August/30

From 2013.igem.org

(Difference between revisions)
(1 - Results of liquid culture of Pfnr-RBS-Amil CP-Term in pSB1C3 in aerobic and anaerobic conditions)
(Notebook : August 30)
Line 20: Line 20:
[[File:PsPfnr3008.jpg|600px]]
[[File:PsPfnr3008.jpg|600px]]
-
In anaerobic condition, the constitutive promoter Pndh* is repressed, RBS_AmilCP-Term isn't expressed. We can't see any coloration of bacterium.  
+
In anaerobic condition, FNR protein (active form) binds to the constitutive promoter Pndh* and repressed ''amilCP'' expression. Therefore, the bacteria have no colour.  
-
In aerobic condition, the constitutive promoter Pndh* isn't repressed, RBS-Amil CP-Term is expressed and we can see bacterium have violet colour.
+
In aerobic condition, FNR protein is inactive and can not bind to constitutive promoter Pndh*, ''amilCP'' is expressed and we can see the bacteria have violet colour.
-
===='''2 - Results of culture of Pfnr with RBS-Amil CP-Term in pSB1C3, NirB with RBS-LacZ-Term in pSB1C3 in aerobic and anaerobic conditions'''====
+
===='''2 - Results of culture of Pfnr with RBS-AmilCP-Term in pSB1C3, PnirB with RBS-LacZ-Term in pSB1C3 in aerobic and anaerobic conditions'''====
XiaoJing
XiaoJing
Line 30: Line 30:
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DE;" |
| style="border:1px solid black;padding:5px;background-color:#DE;" |
-
Purification of 08/29/13 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.
+
Purification of 08/29/13 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 PnirB with RBS-LacZ-Term in pSB1C3 in anaerobic conditions.
|}
|}
Line 36: Line 36:
[[File:PsNirBcult3008.jpg|500px]]
[[File:PsNirBcult3008.jpg|500px]]
-
===='''3 - Ligation of Pfnr with RBS-LacZ-Term in pSB1C3 '''====
+
===='''3 - Ligation of Pndh* with RBS-LacZ-Term in pSB1C3 '''====
XiaoJing  
XiaoJing  
Used quantities :  
Used quantities :  
-
* Pfnr : 8µL
+
* Pndh* : 8µL
* RBS-LacZ-Term in pSB1C3 : 3µL  
* RBS-LacZ-Term in pSB1C3 : 3µL  
* Buffer ligation : 2µL
* Buffer ligation : 2µL
Line 47: Line 47:
* H2O : 6µL
* H2O : 6µL
-
===='''4 - Transformation of ligation of Pfnr with RBS-LacZ-Term in pSB1C3 in DH5α '''====
+
===='''4 - Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB1C3 in DH5α strain '''====
XiaoJing
XiaoJing
Line 53: Line 53:
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
-
We incubate it at 37°C with Xgal in aerobic conditions.
+
We incubate the bacteria in LB-chloramphenicol-Xgal plate at 37°C in aerobic conditions.
-
===='''5 - Ligation of Pfnr with RBS-LacZ-Term in pSB3K3 and NarK with RBS-LacZ-Term in PSB3K3 '''====
+
===='''5 - Ligation of Pfnr with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 '''====
XiaoJing  
XiaoJing  
Used quantities :  
Used quantities :  
-
* Pfnr, NarK : 3µL
+
* Pndh*, PnarK : 3µL
* RBS-LacZ-Term : 3µL
* RBS-LacZ-Term : 3µL
* pSB3K3 : 5µL
* pSB3K3 : 5µL
Line 67: Line 67:
* H2O : 6µL
* H2O : 6µL
-
===='''6 - Transformation of ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and NarK with RBS-LacZ-Term in PSB3K3 in DH5α '''====
+
===='''6 - Transformation of ligation of Pfnr with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 in DH5α strain '''====
XiaoJing
XiaoJing
Line 73: Line 73:
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
-
We incubate it at 37°C with Xgal in anaerobic conditions for NarK with RBS-LacZ-Term in PSB3K3. We incubate it at 37°C with Xgal in aerobic conditions for NarK with Pfnr-LacZ-Term in PSB3K3.
+
We incubate the bacteria at 37°C with LB-Kanamycine-Xgal in anaerobic conditions for PnarK with RBS-LacZ-Term in PSB3K3.
-
===='''7 - Electrophoresis of Colony PCR of Pfnr with RBS_AmilCP-Term  in DH5α '''====
+
We incubate the bacteria at 37°C with LB-Kanamycine-Xgal in aerobic conditions for Pndh* with RBS-LacZ-Term in pSB3K3.
 +
 
 +
===='''7 - Electrophoresis of PCR Colony of Pfnr with RBS_AmilCP-Term  in DH5α strain '''====
XiaoJing
XiaoJing
Line 83: Line 85:
| 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
-
* Well 2 : 20µL of Pfnr with RBS_AmilCP-Term clone 1+4µL of 6X loading dye
+
* Well 2 : 20µL of Pfnr with RBS_AmilCP-Term clone 1 + 4µL of 6X loading dye
-
* Well 3 : 20µL of Pfnr with RBS_AmilCP-Term clone 2+4µL of 6X loading dye     
+
* Well 3 : 20µL of Pfnr with RBS_AmilCP-Term clone 2 + 4µL of 6X loading dye     
-
* Well 4 : 20µL of Pfnr with RBS_AmilCP-Term clone 3+4µL of 6X loading dye   
+
* Well 4 : 20µL of Pfnr with RBS_AmilCP-Term clone 3 + 4µL of 6X loading dye   
-
* Well 5 : 20µL of Pfnr with RBS_AmilCP-Term clone 4+4µL of 6X loading dye   
+
* Well 5 : 20µL of Pfnr with RBS_AmilCP-Term clone 4 + 4µL of 6X loading dye   
* Gel : 1.0%
* Gel : 1.0%
|}
|}
Line 102: Line 104:
===='''Objective : obtaining FNR and BphR2 proteins'''====
===='''Objective : obtaining FNR and BphR2 proteins'''====
-
===='''1 - Electrophoresis of Colony PCR of FNR, RBS-FNR and RBS-BphR2'''====
+
===='''1 - Electrophoresis of PCR Colony of FNR, RBS-FNR and RBS-BphR2'''====
{|
{|
Line 108: Line 110:
| 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
-
* Well 2 to 17 : 5µL of FNR+1µl of 6X loading dye
+
* Well 2 to 17 : 5µL of FNR + 1µl of 6X loading dye
-
* Well 18 to 26 : 5µL of RBS-BphR2+1µl of 6X loading dye
+
* Well 18 to 26 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
* Well 27 : 6µL DNA Ladder
* Well 27 : 6µL DNA Ladder
-
* Well 28 to 41 : 5µL of RBS-FNR+1µl of 6X loading dye
+
* Well 28 to 41 : 5µL of RBS-FNR + 1µl of 6X loading dye
-
* Well 42 to 49 : 5µL of RBS-BphR2+1µl of 6X loading dye
+
* Well 42 to 49 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
* WEll 50 : 6µL DNA Ladder
* WEll 50 : 6µL DNA Ladder
* Gel : 1%
* Gel : 1%

Revision as of 17:37, 4 October 2013

Contents

Notebook : August 30

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Results of liquid culture of Pfnr-RBS-Amil CP-Term in pSB1C3 in aerobic and anaerobic conditions

XiaoJing

IT WORKS !!!

PsPfnr3008.jpg

In anaerobic condition, FNR protein (active form) binds to the constitutive promoter Pndh* and repressed amilCP expression. Therefore, the bacteria have no colour.

In aerobic condition, FNR protein is inactive and can not bind to constitutive promoter Pndh*, amilCP is expressed and we can see the bacteria have violet colour.

2 - Results of culture of Pfnr with RBS-AmilCP-Term in pSB1C3, PnirB with RBS-LacZ-Term in pSB1C3 in aerobic and anaerobic conditions

XiaoJing

Purification of 08/29/13 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 PnirB with RBS-LacZ-Term in pSB1C3 in anaerobic conditions.

PsPfnrcult3008.jpg PsNirBcult3008.jpg

3 - Ligation of Pndh* with RBS-LacZ-Term in pSB1C3

XiaoJing

Used quantities :

  • Pndh* : 8µL
  • RBS-LacZ-Term in pSB1C3 : 3µL
  • Buffer ligation : 2µL
  • Ligase : 1µL
  • H2O : 6µL

4 - Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB1C3 in DH5α strain

XiaoJing

Protocol : Bacterial transformation

We incubate the bacteria in LB-chloramphenicol-Xgal plate at 37°C in aerobic conditions.

5 - Ligation of Pfnr with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3

XiaoJing

Used quantities :

  • Pndh*, PnarK : 3µL
  • RBS-LacZ-Term : 3µL
  • pSB3K3 : 5µL
  • Buffer ligase : 2µL
  • Ligase : 1µL
  • H2O : 6µL

6 - Transformation of ligation of Pfnr with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 in DH5α strain

XiaoJing

Protocol : Bacterial transformation

We incubate the bacteria at 37°C with LB-Kanamycine-Xgal in anaerobic conditions for PnarK with RBS-LacZ-Term in PSB3K3.

We incubate the bacteria at 37°C with LB-Kanamycine-Xgal in aerobic conditions for Pndh* with RBS-LacZ-Term in pSB3K3.

7 - Electrophoresis of PCR Colony of Pfnr with RBS_AmilCP-Term in DH5α strain

XiaoJing

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 20µL of Pfnr with RBS_AmilCP-Term clone 1 + 4µL of 6X loading dye
  • Well 3 : 20µL of Pfnr with RBS_AmilCP-Term clone 2 + 4µL of 6X loading dye
  • Well 4 : 20µL of Pfnr with RBS_AmilCP-Term clone 3 + 4µL of 6X loading dye
  • Well 5 : 20µL of Pfnr with RBS_AmilCP-Term clone 4 + 4µL of 6X loading dye
  • Gel : 1.0%

Expected sizes :

  • Pfnr with RBS_AmilCP-Term : 1000bp

We obtain fragments at the right size.

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

Objective : obtaining FNR and BphR2 proteins

1 - Electrophoresis of PCR Colony of FNR, RBS-FNR and RBS-BphR2

]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 to 17 : 5µL of FNR + 1µl of 6X loading dye
  • Well 18 to 26 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
  • Well 27 : 6µL DNA Ladder
  • Well 28 to 41 : 5µL of RBS-FNR + 1µl of 6X loading dye
  • Well 42 to 49 : 5µL of RBS-BphR2 + 1µl of 6X loading dye
  • WEll 50 : 6µL DNA Ladder
  • Gel : 1%

Expect sizes :

  • FNR : 1096 bp
  • RBS-FNR : 1014 bp
  • RBS-BphR2 : 1469 bp

We obtain fragment at right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.


Previous day Back to calendar Next day