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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 !!!

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.

**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.

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@@ Line 20: / Line 20: @@
 [[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
@@ Line 30: / Line 30: @@
 {|
 | 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]]
-===='''3 - Ligation of Pfnr with RBS-LacZ-Term in pSB1C3 '''====
+===='''3 - Ligation of Pndh* with RBS-LacZ-Term in pSB1C3 '''====
 XiaoJing
 Used quantities :
-* Pfnr : 8µL
+* Pndh* : 8µL
 * RBS-LacZ-Term in pSB1C3 : 3µL
 * Buffer ligation : 2µL
@@ Line 47: / Line 47: @@
 * 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
@@ Line 53: / Line 53: @@
 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
 Used quantities :
-* Pfnr, NarK : 3µL
+* Pndh*, PnarK : 3µL
 * RBS-LacZ-Term : 3µL
 * pSB3K3 : 5µL
@@ Line 67: / Line 67: @@
 * 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
@@ Line 73: / Line 73: @@
 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
@@ Line 83: / Line 85: @@
 | style="width:350px;border:1px solid black;vertical-align:top;" |
 * 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%
 |}
@@ Line 102: / Line 104: @@
 ===='''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;" |
 * 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 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
 * Gel : 1%

Team:Paris Saclay/Notebook/August/30

From 2013.igem.org

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

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

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

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

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

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

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

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