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Notebook : August 26

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI

XiaoJing

BBa_K1155000 :
- DNA : 14µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL

BBa_K1155004 clone 6 :
- DNA : 14µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL

BBa_K1155004 clone 7 :
- DNA : 14µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL

BBa_K1155005 clone 6 :
- DNA : 7µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 7µL

BBa_K1155005 clone 7 :
- DNA : 14µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL

BBa_K1155006 clone 6 :
- DNA : 4µL
- SpeI FD : 2µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 10µL

BBa_K1155004 already digested by SpeI :
- DNA : 13µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 3µL

BBa_K1155005 already digested by SpeI :
- DNA : 10µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 6µL

BBa_K1155006 already digested by SpeI :
- DNA : 9µL
- PstI FD : 2µL
- Buffer FD : 2µL
- H2O : 7µL

We incubate the digestion for 30 minutes at 37°C.

2 - Inactivation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

XiaoJing

Protocol : Ethanol precipitation

We used 20µL of DNA.

Nanodrop :

Pndh* : 91.8ng/µL
NarK clone 6 : 19.8ng/µL
Nark already digested by SpeI : 15.1ng/µL
NarG clone 6 : 13.4ng/µL
NarG clone 7 : 103.9ng/µL
narG already digested by SpeI : 17.4ng/µL
NirB clone 6 : 46ng/µL
NirB clone 7 : 61.6ng/µL
NirB already digested by SpeI : 16.1ng/µL

According to the result of the Nanodrop, we decide to do ligations with Pndh* and nirB clone 6.

**3 - Ligation of Pndh*, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3**

XiaoJing

Used quantities :

NirB clone 6 with RBS-LacZ or RBS-Amil CP, Pndh* with RBS-LacZ or RBS-Amil CP :

RBS-LacZ-Term or RBS-Amil CP-Term : 3µL
Pndh* or NirB clone 6 : 5µL
Buffer ligase : 2µL
Ligase T4 : 2µL
H20 : 8µL

We incubate the ligation for 1h at 37°C.

4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

XiaoJing

Protocol : Bacterial transformation

5- Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20

XioaJing

Transformation of 08/19/13 works. We will purify our colonies.

We streaked colonies into ampicilin and chloramphenicol plates and incubate at 30°C.

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

Objective : obtaining FNR and BphR2

1 - Gibson assembly

XiaoJing

Tranformation of 08/21 didn't work. We will do the Gibson assembly again.

Used quantities :

RBS-BphR2 :
- PSB1C3 : 3µL
- BphR2 Part I : 1µL
- BphR2 Part II : 1µL
- Gibson mix : 15µL

FNR :
- PSB1C3 : 3µL
- FNR Part I : 1µL
- FNR Part II : 1µL
- Gisbon mix : 15µL

RBS-FNR :
- PSB1C3 : 3µL
- RBS-FNR Part I : 1µL
- FNR Part II : 1µL
- Gibson mix : 15µL

We keep these mix at 50°C for 1h.

2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α

XiaoJing

Protocol : Bacterial transformation

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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 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI/PstI'''====
+===='''1 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI'''====
 XiaoJing
-* Bba_K1155000 :
+* BBa_K1155000 :
 ** DNA : 14µL
 ** SpeI FD : 2µL
@@ Line 19: / Line 19: @@
 ** Buffer FD : 2µL
-* Bba_K1155004 clone 6 :
+* BBa_K1155004 clone 6 :
 ** DNA : 14µL
 ** SpeI FD : 2µL
@@ Line 25: / Line 25: @@
 ** Buffer FD : 2µL
-* Bba_K1155004 clone 7 :
+* BBa_K1155004 clone 7 :
 ** DNA : 14µL
 ** SpeI FD : 2µL
@@ Line 31: / Line 31: @@
 ** Buffer FD : 2µL
-* Bba_K1155005 clone 6 :
+* BBa_K1155005 clone 6 :
 ** DNA : 7µL
 ** SpeI FD : 2µL
@@ Line 38: / Line 38: @@
 ** H2O : 7µL
-* Bba_K1155005 clone 7 :
+* BBa_K1155005 clone 7 :
 ** DNA : 14µL
 ** SpeI FD : 2µL
@@ Line 44: / Line 44: @@
 ** Buffer FD : 2µL
-* Bba_K1155006 clone 6 :
+* BBa_K1155006 clone 6 :
 ** DNA : 4µL
 ** SpeI FD : 2µL
@@ Line 51: / Line 51: @@
 ** H2O : 10µL
-Bba_K1155004 already digested by SpeI :
+* BBa_K1155004 already digested by SpeI :
 ** DNA : 13µL
 ** PstI FD : 2µL
@@ Line 57: / Line 57: @@
 ** H2O : 3µL
-Bba_K1155005 already digested by SpeI :
+* BBa_K1155005 already digested by SpeI :
 ** DNA : 10µL
 ** PstI FD : 2µL
@@ Line 63: / Line 63: @@
 ** H2O : 6µL
-* Bba_K1155006 already digested by SpeI :
+* BBa_K1155006 already digested by SpeI :
 ** DNA : 9µL
 ** PstI FD : 2µL
@@ Line 69: / Line 69: @@
 ** H2O : 7µL
-We keep digestions for 30 minutes at 37°C.
+We incubate the digestion for 30 minutes at 37°C.
-===='''2 - Denaturation of SpeI/PstI used for the digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+===='''2 - Inactivation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
 XiaoJing
@@ Line 80: / Line 80: @@
 Nanodrop :
-* Pfnr : 91.8ng/µL
+* Pndh* : 91.8ng/µL
 * NarK clone 6 : 19.8ng/µL
 * Nark already digested by SpeI : 15.1ng/µL
@@ Line 92: / Line 92: @@
 {|
 | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-According to the result of the Nanodrop, we decide to do ligations with Pfnr and nirB clone 6.
+According to the result of the Nanodrop, we decide to do ligations with Pndh* and nirB clone 6.
 |}
-===='''3 - Ligation of Pfnr, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3'''====
+===='''3 - Ligation of Pndh*, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3'''====
 XiaoJing
@@ Line 101: / Line 101: @@
 Used quantities :
-NirB clone 6 with RBS-LacZ or RBS-Amil CP, Pfnr with RBS-LacZ or RBS-Amil CP :
+NirB clone 6 with RBS-LacZ or RBS-Amil CP, Pndh* with RBS-LacZ or RBS-Amil CP :
 * RBS-LacZ-Term or RBS-Amil CP-Term : 3µL
-* Pfnr or NirB clone 6 : 5µL
+* Pndh* or NirB clone 6 : 5µL
 * Buffer ligase : 2µL
 * Ligase T4 : 2µL
 * H20 : 8µL
-We keep the ligation for 1h at 37°C.
+We incubate the ligation for 1h at 37°C.
 ===='''4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3'''====
@@ Line 115: / Line 115: @@
 Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
+===='''5- Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20'''====
+XioaJing
+{|
+| style="border:1px solid black;padding:5px;background-color:#DE;" |
+Transformation of 08/19/13 works. We will purify our colonies.
+|}
+We streaked colonies into ampicilin and chloramphenicol plates and incubate at 30°C.
 ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===

Team:Paris Saclay/Notebook/August/26

From 2013.igem.org

Latest revision as of 22:16, 4 October 2013

Contents

Notebook : August 26

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006 by SpeI/PstI

2 - Inactivation of SpeI/PstI used for the digestion of BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

**3 - Ligation of Pndh*, NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3**

4 - Transformation of ligation of Pfnr and NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3

5- Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20

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

Objective : obtaining FNR and BphR2

1 - Gibson assembly

2 - Transformation of FNR, RBS-FNR and RBS-BphR2 in DH5α