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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 Pndh*-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 Pndh* 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 Pndh* 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 Pndh* with RBS_AmilCP-Term in DH5α strain**

XiaoJing

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

Expected sizes :

Pndh* with RBS_AmilCP-Term : 1000bp

We obtained fragments at the right size.

8 - Result of the purification colony of MG1655Z1 Δfnr

XiaoJing

It works. Our plasmid didn't contain any antibiotic resistance gene. Colonies grew in LB medium and didn't grow on kanamycin, ampicilin, chloramphenicol medium. We obtain strain MG1655Z1 Δfnr.

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 obtained fragments at the right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.

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@@ Line 7: / Line 7: @@
 ==='''A - Aerobic/Anaerobic regulation system'''===
-===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+===='''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'''====
+===='''1 - Results of liquid culture of Pndh*-RBS-Amil CP-Term in pSB1C3 in aerobic and anaerobic conditions'''====
 XiaoJing
@@ Line 20: / Line 20: @@
 [[File:PsPfnr3008.jpg|600px]]
-In anaerobic conditions, thank to promotor Pfnr, RBS-Amil CP-Term gene is activated and we can see a violet coloration of bacterias.
+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 conditions, promotor Pfnr is inactivated, RBS-Amil CP-Term isn't activated and we can't see any coloration of bacterias.
+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-RBS-Amil CP-Term in PSB1C3, NirB with RBS-LacZ-Term in PSB1C3 in aerobic and anaerobic conditions'''====
+===='''2 - Results of culture of Pndh* 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
+* RBS-LacZ-Term in pSB1C3 : 3µL
 * Buffer ligation : 2µL
 * Ligase : 1µL
 * 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.
-===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''====
+===='''5 - Ligation of Pfnr with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 '''====
-===='''1 - Ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and NarK with RBS-LacZ-Term in PSB3K3 '''====
 XiaoJing
 Used quantities :
-* Pfnr, NarK : 3µL
+* Pndh*, PnarK : 3µL
 * RBS-LacZ-Term : 3µL
-* PSB3K3 : 5µL
+* pSB3K3 : 5µL
 * Buffer ligase : 2µL
 * Ligase : 1µL
 * H2O : 6µL
-===='''2 - 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 Pndh* with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 in DH5α strain '''====
 XiaoJing
@@ Line 75: / 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.
+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 Pndh* with RBS_AmilCP-Term  in DH5α strain '''====
+XiaoJing
+{|
+| style="width:350px;border:1px solid black;" | [[File:Psgel13008.jpg]]
+| style="width:350px;border:1px solid black;vertical-align:top;" |
+* Well 1 : 6µL DNA Ladder
+* Well 2 : 20µL of Pndh* with RBS_AmilCP-Term clone 1 + 4µL of 6X loading dye
+* Well 3 : 20µL of Pndh* with RBS_AmilCP-Term clone 2 + 4µL of 6X loading dye
+* Well 4 : 20µL of Pndh* with RBS_AmilCP-Term clone 3 + 4µL of 6X loading dye
+* Well 5 : 20µL of Pndh* with RBS_AmilCP-Term clone 4 + 4µL of 6X loading dye
+* Gel : 1.0%
+|}
+Expected sizes :
+* Pndh* with RBS_AmilCP-Term : 1000bp
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We obtained fragments at the right size.
+|}
+===='''8 - Result of the purification colony of MG1655Z1 Δfnr'''====
+XiaoJing
+{|
+| style="border:1px solid black;padding:5px;background-color:#DE;" |
+It works. Our plasmid didn't contain any antibiotic resistance gene. Colonies grew in LB medium and didn't grow on kanamycin, ampicilin, chloramphenicol medium. We obtain strain MG1655Z1 Δfnr.
+|}
+[[File:PsLB3008.jpg|311px]][[File:PsKm3008.jpg|300px]]
+[[File:PsCm3008.jpg|311px]][[File:PsAm3008.jpg|311px]]
 ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
@@ Line 81: / Line 116: @@
 ===='''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'''====
 {|
-| style="width:350px;border:1px solid black;" |]]
+| style="width:350px;border:1px solid black;" |[[File:Psgel23008.jpg]]
 | 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
+* Well 50 : 6µL DNA Ladder
 * Gel : 1%
 |}
@@ Line 103: / Line 138: @@
 {|
 | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-We obtain fragment at right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.
+We obtained fragments at the right size for FNR and RBS-FNR. The Gisbon assembly of 08/26/13 was good.
 |}

Team:Paris Saclay/Notebook/August/30

From 2013.igem.org

Latest revision as of 01:23, 5 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 Pndh*-RBS-Amil CP-Term in pSB1C3 in aerobic and anaerobic conditions**

**2 - Results of culture of Pndh* 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 Pndh* with RBS-LacZ-Term in pSB3K3 and PnarK with RBS-LacZ-Term in pSB3K3 in DH5α strain**

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

8 - Result of the purification colony of MG1655Z1 Δfnr

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