Team:Toulouse/Notebook/project-monitoring

From 2014.igem.org

(Difference between revisions)
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-
Fungicides
+
<br>Fungicides
-
D4E1
+
<br>D4E1
-
1. Amplification of synthetic gene (D4E1 on pEX-A2)
+
<br>1. Amplification of synthetic gene (D4E1 on pEX-A2)
-
• Transformation of D4E1 (pEX-A2) into E. coli
+
<br>• Transformation of D4E1 (pEX-A2) into E. coli
-
Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
+
<br>Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
-
Concentration of D4E1 : 115ng/µL
+
<br>Concentration of D4E1 : 115ng/µL
-
Date: 07/21//2014
+
<br>Date: 07/21//2014
-
Result: We obtained distinct colonies on LA + Ampicillin (100 µg/mL  )
+
<br>Result: We obtained distinct colonies on LA + Ampicillin (100 µg/mL  )
-
• Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli
+
<br>• Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli
-
Date: 07/22/2014
+
<br>Date: 07/22/2014
-
Result : Culture of 4 clones ok
+
<br>Result : Culture of 4 clones ok
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli
+
<br>• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli
-
Buffer EB at 50-55°C
+
<br>Buffer EB at 50-55°C
-
Date: 07/23/2014
+
<br>Date: 07/23/2014
-
• Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
+
<br>• Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
-
Date: 07/23/2014
+
<br>Date: 07/23/2014
-
Result : 4*20µL D4E1 digested with EcoRI and PstI  all the clones seem to have the right D4E1 gene.
+
<br>Result : 4*20µL D4E1 digested with EcoRI and PstI  all the clones seem to have the right D4E1 gene.
   
   
-
2. Cloning D4E1 in pSB1C3
+
<br>2. Cloning D4E1 in pSB1C3
-
• Digestion of D4E1 on pEX-A2 and pSB1C3
+
<br>• Digestion of D4E1 on pEX-A2 and pSB1C3
-
Date: 07/23/2014
+
<br>Date: 07/23/2014
-
• Ligation of D4E1 in pSB1C3
+
<br>• Ligation of D4E1 in pSB1C3
-
Date: 07/23/2014
+
<br>Date: 07/23/2014
-
• Transformation in E.coli
+
<br>• Transformation in E.coli
-
Date : 07/23/2014
+
<br>Date : 07/23/2014
-
• Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed intoE. coli
+
<br>• Culture of 6 clones: A, B, C, D, E, F of D4E1 (pSB1C3) transformed intoE. coli
-
Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies
+
<br>Processing details: 5mL of LB + chloramphenicol (15µg/mL) , using sterile plastic loop to culture colonies
-
Finished by: Emeline and Diane
+
<br>Finished by: Emeline and Diane
-
Dated: 07/24/2014
+
<br>Dated: 07/24/2014
-
Result: Culture of 4 clones ok
+
<br>Result: Culture of 4 clones ok
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
+
<br>• QIAprep Spin Miniprep Kit Using a Microcentrifuge
-
Date: 07/25/2014
+
<br>Date: 07/25/2014
-
• Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis
+
<br>• Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis
-
Date: 07/25/2014
+
<br>Date: 07/25/2014
-
Result: clones C, D have the expected construction
+
<br>Result: clones C, D have the expected construction
-
• PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
+
<br>• PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
-
Date: 07/25/2014
+
<br>Date: 07/25/2014
-
Result: clones C, D have the expected construction, and placed in cryopreservation.
+
<br>Result: clones C, D have the expected construction, and placed in cryopreservation.
-
3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)
+
<br>3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)
-
• Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)
+
<br>• Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)
-
Finished by: Emeline and Diane
+
<br>Finished by: Emeline and Diane
-
Dated: 07/24/2014
+
<br>Dated: 07/24/2014
-
Result: 20µL digestion of D4E1 on pEX-A2 and of K823003
+
<br>Result: 20µL digestion of D4E1 on pEX-A2 and of K823003
-
• Ligation of D4E1 in K823003 (Pveg on pSB1C3)
+
<br>• Ligation of D4E1 in K823003 (Pveg on pSB1C3)
-
Date: 07/24/2014
+
<br>Date: 07/24/2014
-
Result: 20µL ligation of D4E1 in K823003
+
<br>Result: 20µL ligation of D4E1 in K823003
-
• Transformation of ligation products in E.coli
+
<br>• Transformation of ligation products in E.coli
-
Date : 07/24/2014
+
<br>Date : 07/24/2014
-
Result: E.coli transformed by D4E1+K823003
+
<br>Result: E.coli transformed by D4E1+K823003
-
• Culture of 6 clones: A, B, C, D, E, F of transformed E. coli
+
<br>• Culture of 6 clones: A, B, C, D, E, F of transformed E. coli
-
Date: 07/26/2014
+
<br>Date: 07/26/2014
-
Result: Culture of 4 clones ok
+
<br>Result: Culture of 4 clones ok
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
+
<br>• QIAprep Spin Miniprep Kit Using a Microcentrifuge
-
Date: 07/28/2014
+
<br>Date: 07/28/2014
-
• PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
+
<br>• PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
-
Dated : 07/28/2014
+
<br>Dated : 07/28/2014
-
Result : clones E, F seem to have the expected construction
+
<br>Result : clones E, F seem to have the expected construction
-
• Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis
+
<br>• Digestion of clones E, F of D4E1+K823003 (Pveg on pSB1C3) with EcoRI and PstI + electrophoresis
-
Date: 28/07/2014
+
<br>Date: 28/07/2014
-
Result : clones C, D have the expected construction and are placed in cryopreservation.
+
<br>Result : clones C, D have the expected construction and are placed in cryopreservation.
   
   
-
4. Cloning Pveg+D4E1 on pSBBS4S (K823022)  
+
<br>4. Cloning Pveg+D4E1 on pSBBS4S (K823022)  
-
Date: 08/13/2014
+
<br>Date: 08/13/2014
   
   
-
5. Cloning Pveg + D4E1 on pSBBS1C lacZ (23)
+
<br>5. Cloning Pveg + D4E1 on pSBBS1C lacZ (23)
-
COMPLETER !
+
<br>GAFP1
 +
<br>1. Amplification of synthetic gene (GAFP1 on pEX-A2)
 +
<br>• Transformation of GAFP1 (pEX-A2) into E.coli
 +
<br>Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
 +
<br>Concentration of GAFP1 : 145ng/µL
 +
<br>Date : 07/21/2014
 +
<br>Result: We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)
 +
<br>• Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli
 +
<br>Date: 07/22/2014
 +
<br>Result: Culture of 4 clones ok
 +
<br>• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli
 +
<br>Date: 07/23/2014
 +
<br>• Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel <br>Electrophoresis
 +
<br>Date: 07/23/201
-
 
+
<br>2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli
-
 
+
<br>• Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSB1C3)
-
 
+
<br>Date: 07/23/2014
-
 
+
<br>Result : BBa_K606013 : 860 bp
-
 
+
<br> We decide to conserve the miniprep B for BBa_K606013
-
 
+
<br>• Ligation GAFP1 and BBa_K606013
-
GAFP1
+
<br>Date : 07/23/2014
-
1. Amplification of synthetic gene (GAFP1 on pEX-A2)
+
<br>• Tranformation of ligation products into E. coli
-
• Transformation of GAFP1 (pEX-A2) into E.coli
+
<br>Date : 07/23/2014
-
Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
+
<br>• Culture of x clones of GAFP1+K606013 in E. coli
-
Concentration of GAFP1 : 145ng/µL
+
<br>Date : 07/24/2014
-
Date : 07/21/2014
+
<br>• QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into E. coli
-
Result: We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)
+
<br>Date: 07/25/2014
-
• Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli
+
<br>• PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis
-
Date: 07/22/2014
+
<br>Date: 07/25/2014
-
Result: Culture of 4 clones ok
+
<br>Result : clones A, C, D, F seem to have the right construction
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli
+
<br>• Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis
-
Date: 07/23/2014
+
<br>Date: 07/25/2014
-
• Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis
+
-
Date: 07/23/201
+
-
 
+
-
2. Cloning GAFP1 gene on PSB1C3 = K1364002 in E. coli
+
-
• Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSB1C3)
+
-
Date: 07/23/2014
+
-
Result : BBa_K606013 : 860 bp
+
-
 We decide to conserve the miniprep B for BBa_K606013
+
-
• Ligation GAFP1 and BBa_K606013
+
-
Date : 07/23/2014
+
-
• Tranformation of ligation products into E. coli
+
-
Date : 07/23/2014
+
-
• Culture of x clones of GAFP1+K606013 in E. coli
+
-
Date : 07/24/2014
+
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge: 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) into E. coli
+
-
Date: 07/25/2014
+
-
• PCR on 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) + electrophoresis
+
-
Date: 07/25/2014
+
-
Result : clones A, C, D, F seem to have the right construction
+
-
• Digestion of 6 clones of GAFP1+K606013 = K1364002 (pSB1C3) by EcoR1 and Pst1 + electrophoresis
+
-
Date: 07/25/2014
+
   
   
-
3.  Cloning GAFP1+terminator(B0015) = K1364007 (pSB1C3)
+
<br>3.  Cloning GAFP1+terminator(B0015) = K1364007 (pSB1C3)
-
• Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)
+
<br>• Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)
-
Date : 07/24/2014
+
<br>Date : 07/24/2014
-
• Ligation of GAFP1 in B0015 (terminator on pSB1C3)
+
<br>• Ligation of GAFP1 in B0015 (terminator on pSB1C3)
-
Date: 07/24/2014
+
<br>Date: 07/24/2014
-
• Transformation of ligation products in E.coli
+
<br>• Transformation of ligation products in E.coli
-
Date: 07/24/2014
+
<br>Date: 07/24/2014
-
• Culture of 6 clones: A, B, C, D, E, F transformed in E. coli
+
<br>• Culture of 6 clones: A, B, C, D, E, F transformed in E. coli
-
Date: 07/26/2014
+
<br>Date: 07/26/2014
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
+
<br>• QIAprep Spin Miniprep Kit Using a Microcentrifuge
-
Date : 07/28/2014
+
<br>Date : 07/28/2014
-
• PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis
+
<br>• PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis
-
Date: 07/28/2014
+
<br>Date: 07/28/2014
-
Result : clones B, C, D, E, F seem to have the expected construction.
+
<br>Result : clones B, C, D, E, F seem to have the expected construction.
-
• Digestion of clones E, F of GAFP1+Ter B0015 = K1364007 with EcoRI and PstI + electrophoresis
+
<br>• Digestion of clones E, F of GAFP1+Ter B0015 = K1364007 with EcoRI and PstI + electrophoresis
-
Date: 07/28/2014
+
<br>Date: 07/28/2014
-
Result : clones B, C, D, E, F have the expected construction.
+
<br>Result : clones B, C, D, E, F have the expected construction.
   
   
-
4. Cloning GAFP1+terminator with Pveg on pSB1C3 (K1364008) in E. coli
+
<br>4. Cloning GAFP1+terminator with Pveg on pSB1C3 (K1364008) in E. coli
-
• Digestion of GAFP1+ter = K1364007 on pSB1C3 and K823003 (terminator on pSB1C3) + electrophoresis
+
<br>• Digestion of GAFP1+ter = K1364007 on pSB1C3 and K823003 (terminator on pSB1C3) + electrophoresis
-
Date: 07/29/2014
+
<br>Date: 07/29/2014
-
• Gel extraction of K1364007 (extraction of GAFP1+ter gene)
+
<br>• Gel extraction of K1364007 (extraction of GAFP1+ter gene)
-
Date: 07/29/2014
+
<br>Date: 07/29/2014
-
• Ligation of GAFP1+ter in K823003 (Pveg on pSB1C3)
+
<br>• Ligation of GAFP1+ter in K823003 (Pveg on pSB1C3)
-
Date : 07/29/2014
+
<br>Date : 07/29/2014
-
Result : 20µL ligation of GAFP1+ter in K823003
+
<br>Result : 20µL ligation of GAFP1+ter in K823003
-
• Transformation of ligation products in E.coli
+
<br>• Transformation of ligation products in E.coli
-
Date: 07/29/2014
+
<br>Date: 07/29/2014
-
• Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli
+
<br>• Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli
-
Date: 07/30/2014
+
<br>Date: 07/30/2014
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
+
<br>• QIAprep Spin Miniprep Kit Using a Microcentrifuge
-
Date : 07/31/2014
+
<br>Date : 07/31/2014
-
• PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis
+
<br>• PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis
-
   Date: 31/07/2014
+
   <br>Date: 31/07/2014
-
Result : clones A, B, C, D, E, G, H seem to have the expected construction
+
<br>Result : clones A, B, C, D, E, G, H seem to have the expected construction
-
• Digestion of clones A, B, C, D, E, G, H of Pveg+K1364007 = K1364008 with EcoRI and PstI + electrophoresis
+
<br>• Digestion of clones A, B, C, D, E, G, H of Pveg+K1364007 = K1364008 with EcoRI and PstI + electrophoresis
-
Date: 31/07/2014
+
<br>Date: 31/07/2014
-
Result : clones A, B, C, D, E, G, H have the expected construction
+
<br>Result : clones A, B, C, D, E, G, H have the expected construction
   
   
-
5.  Cloning Pveg+GAFP1+Ter B0015 (BBa_K1364008) on pSBBS4S (BBa_K823022)
+
<br>5.  Cloning Pveg+GAFP1+Ter B0015 (BBa_K1364008) on pSBBS4S (BBa_K823022)
-
• Digestion of Pveg+GAFP1+ ter B0015 (K1364008) on pSB1C3 and PsBBs4S (K823022)
+
<br>• Digestion of Pveg+GAFP1+ ter B0015 (K1364008) on pSB1C3 and PsBBs4S (K823022)
-
Date: 08/01/2014
+
<br>Date: 08/01/2014
-
• Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)
+
<br>• Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)
-
Date: 08/01/2014
+
<br>Date: 08/01/2014
-
• Transformation of ligation products in E.coli
+
<br>• Transformation of ligation products in E.coli
-
Date : 08/01/2014
+
<br>Date : 08/01/2014
-
• Culture of  8 clones: A, B, C, D, E, F, G, H of transformed E. coli
+
<br>• Culture of  8 clones: A, B, C, D, E, F, G, H of transformed E. coli
-
Date: 08/02/2014
+
<br>Date: 08/02/2014
-
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
+
<br>• QIAprep Spin Miniprep Kit Using a Microcentrifuge
-
Date: 08/04/2014
+
<br>Date: 08/04/2014
-
• Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis
+
<br>• Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis
-
Date: 08/04/2014
+
<br>Date: 08/04/2014
-
Result: clones A, E, F have the right construction
+
<br>Result: clones A, E, F have the right construction
-
6. Cloning GAFP1+Ter B0015 on psBBs1C lacZ (23)
 
-
 
-
COMPLETER
 
-
 
-
7. Tests
 
-
COMPLETER
 
-
EcAMP
 
-
The EcAMP part was sent by the Utah iGEM team. It was on a pUC plasmid (ampicilin resistance) with biobrick suffix and prefix.
 
-
1. Transformation of EcAMP in Escherichia coli MC 1061
 
-
Date: 07/25/2014
 
-
2. Spreading of coli cells transformed with pUC + Utah
+
<br>EcAMP
-
Date: 07/28/2014
+
<br>The EcAMP part was sent by the Utah iGEM team. It was on a pUC plasmid (ampicilin resistance) with biobrick suffix and prefix.
 +
<br>1. Transformation of EcAMP in Escherichia coli MC 1061
 +
<br>Date: 07/25/2014
-
3. Liquid culture + Miniprep + Test of the miniprep
+
<br>2. Spreading of coli cells transformed with pUC + Utah
-
Date: 07/30/2014
+
<br>Date: 07/28/2014
-
4. Cloning 1: EcAMP + Pveg + RBS
+
<br>3. Liquid culture + Miniprep + Test of the miniprep
-
• Digestion of EcAMP (INSERT) by XbaI and PstI
+
<br>Date: 07/30/2014
 +
 
 +
<br>4. Cloning 1: EcAMP + Pveg + RBS
 +
<br>• Digestion of EcAMP (INSERT) by XbaI and PstI
Date: 07/31/2014
Date: 07/31/2014
-
• Digestion of Pveg + RBS (VECTOR)
+
<br>• Digestion of Pveg + RBS (VECTOR)
-
  Date: 07/31/2014
+
  <br> Date: 07/31/2014
-
• Ligation and transformation
+
<br>• Ligation and transformation
-
Date: 08/04/2014
+
<br>Date: 08/04/2014
-
• PCR test
+
<br>• PCR test
Date: 08/05/2014
Date: 08/05/2014
-
• Analytical digestion
+
<br>• Analytical digestion
-
Date: 08/05/2014
+
<br>Date: 08/05/2014
-
 
+
-
 The first cloning show a lack of DNA in the Miniprep EcAMP. The bands are hardly visible on the gel for the linearization of EcAMP + Pveg + RBS. The problem came from the PCR cleanup step: the fragment size of EcAMP is lower than 200pb.
+
-
 
+
-
5. Cloning 2: EcAMP + Pveg + RBS
+
-
Date: 06/08/2014
+
-
• Digestion of EcAMP (INSERT) by XbaI and PstI
+
-
• Digestion of Pveg + RBS (VECTOR)
+
-
• Heat inactivation of the enzymes
+
-
• Ligation and transformation
+
-
• PCR test
+
-
Date: 07/08/2014
+
-
 
+
-
 
+
-
• Striation on a petri dish to purify the clone
+
-
Purpose: to isolate a clone with vector+insert
+
-
Date: 08/072014
+
-
 
+
-
• Miniprep of Pveg+SpoVG+EcAMP and analytic digestion
+
-
Date: 08/08/2014
+
-
 
+
-
• Ligation of Pveg SpoVG EcAMP with double terminateur B0015 +Transformation and liquid culture
+
-
Date: 08/11/2014
+
-
 
+
-
• Miniprep of Pveg SpoVG EcAMP + analytic digestion
+
-
Date : 08/13/2014
+
-
 
+
-
• Cloning K1364011 (EcAMP+Pveg +SpoVG + B0015) + K823022 (psBbs4S) : Digestion, ligation, transformation
+
-
Date: 08/19/2014
+
-
 
+
-
• Cloning K1364011 + K823023 (pSBBS1C) : Digestion, ligation, transformation
+
-
Date: 08/18/2014
+
-
 
+
-
• Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test
+
-
Date: 08/21/2014
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
 +
<br> The first cloning show a lack of DNA in the Miniprep EcAMP. The bands are hardly visible on the gel for the linearization of EcAMP + Pveg + RBS. The problem came from the PCR cleanup step: the fragment size of EcAMP is lower than 200pb.
 +
<br>5. Cloning 2: EcAMP + Pveg + RBS
 +
<br>Date: 06/08/2014
 +
<br>• Digestion of EcAMP (INSERT) by XbaI and PstI
 +
<br>• Digestion of Pveg + RBS (VECTOR)
 +
<br>• Heat inactivation of the enzymes
 +
<br>• Ligation and transformation
 +
<br>• PCR test
 +
<br>Date: 07/08/2014
 +
<br>• Striation on a petri dish to purify the clone
 +
<br>Purpose: to isolate a clone with vector+insert
 +
<br>Date: 08/072014
 +
<br>• Miniprep of Pveg+SpoVG+EcAMP and analytic digestion
 +
<br>Date: 08/08/2014
 +
<br>• Ligation of Pveg SpoVG EcAMP with double terminateur B0015 +Transformation and liquid culture
 +
<br>Date: 08/11/2014
 +
<br>• Miniprep of Pveg SpoVG EcAMP + analytic digestion
 +
<br>Date : 08/13/2014
 +
<br>• Cloning K1364011 (EcAMP+Pveg +SpoVG + B0015) + K823022 (psBbs4S) : Digestion, ligation, transformation
 +
<br>Date: 08/19/2014
 +
<br>• Cloning K1364011 + K823023 (pSBBS1C) : Digestion, ligation, transformation
 +
<br>Date: 08/18/2014
 +
<br>• Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test
 +
<br>Date: 08/21/2014
-
Assembling the fungicides module
+
<br>Assembling the fungicides module
-
D4E1 + GAFP1  
+
<br>D4E1 + GAFP1  
-
1.  Cloning GAFP1+D4E1 on pSB1C3: BBa_K1364012  
+
<br>1.  Cloning GAFP1+D4E1 on pSB1C3: BBa_K1364012  
-
• Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3
+
<br>• Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3
-
  Date: 07/28/2014
+
  <br>Date: 07/28/2014
-
Result : We obtained 40µL digestion of D4E1 on pEX-A2 and of GAFP1 on pSB1C3.
+
<br>Result : We obtained 40µL digestion of D4E1 on pEX-A2 and of GAFP1 on pSB1C3.
-
• Ligation of digestions of D4E1 on pEX-A2 and of GAFP1 on pSB1C3
+
<br>• Ligation of digestions of D4E1 on pEX-A2 and of GAFP1 on pSB1C3
-
Date: 07/28/2014
+
<br>Date: 07/28/2014
-
• Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli
+
<br>• Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli
-
Date: 07/28/2014
+
<br>Date: 07/28/2014
   
   
-
• PCR of GAFP1+D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
+
<br>• PCR of GAFP1+D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
-
Date: 07/29/2014
+
<br>Date: 07/29/2014
-
Result: clones A, C, F, G seem to have the expected construction.
+
<br>Result: clones A, C, F, G seem to have the expected construction.
   
   
-
• Digestion of GAFP1+D4E1 (pSB1C3) A, C, F, G + electrophoresis
+
<br>• Digestion of GAFP1+D4E1 (pSB1C3) A, C, F, G + electrophoresis
-
Date: 07/30/2014
+
<br>Date: 07/30/2014
-
Result: clone  F (BBa_K1364012) has the expected construction and is placed on cryopreservation.
+
<br>Result: clone  F (BBa_K1364012) has the expected construction and is placed on cryopreservation.
   
   
-
2. Cloning GAFP1+D4E1 (BBa_K1364012) on Pveg plasmid (BBa_K823003): BBa_K1364013
+
<br>2. Cloning GAFP1+D4E1 (BBa_K1364012) on Pveg plasmid (BBa_K823003): BBa_K1364013
-
3. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS4S (K823022)
+
<br>3. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS4S (K823022)
-
4. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS1C lacZ (K823023)
+
<br>4. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS1C lacZ (K823023)
-
5. Fungicides tests
+
<br>5. Fungicides tests
-
Cloning D4E1-GAFP1-EcAMP: BBa_K1364014
+
<br>Cloning D4E1-GAFP1-EcAMP: BBa_K1364014
-
1.  Construction of BBa_K1364014 in PsB1C3, in E. coli
+
<br>1.  Construction of BBa_K1364014 in PsB1C3, in E. coli
-
• Digestion of K1364010 and K1364012
+
<br>• Digestion of K1364010 and K1364012
-
Digestion of K13664010 by Spe1 and Pst1, and digestion of K1364012 by XbaI and Pst1.
+
<br>Digestion of K13664010 by Spe1 and Pst1, and digestion of K1364012 by XbaI and Pst1.
-
Date: 08/11/2014
+
<br>Date: 08/11/2014
-
Result : We obtained digested fragments of K1364012 and ~500 bp fragment of K1364010
+
<br>Result : We obtained digested fragments of K1364012 and ~500 bp fragment of K1364010
-
• Ligation of ~500 bp fragment from K1364010 and K1364012
+
<br>• Ligation of ~500 bp fragment from K1364010 and K1364012
-
Date: 08/11/2014
+
<br>Date: 08/11/2014
-
Result: We obtained ligation of K1364012 and ~500 bp fragment of K1364010
+
<br>Result: We obtained ligation of K1364012 and ~500 bp fragment of K1364010
-
• Transformation of ligation of ~500 bp fragment from K1364010 and K1364012 = K1364014 in E.coli
+
<br>• Transformation of ligation of ~500 bp fragment from K1364010 and K1364012 = K1364014 in E.coli
-
Date: 08/12/2014
+
<br>Date: 08/12/2014
-
• Testing 8 E.coli+K1364014 clones
+
<br>• Testing 8 E.coli+K1364014 clones
-
Date: 08/14/2014  
+
<br>Date: 08/14/2014  
-
• Testing 15 E.coli+K1364014 clones
+
<br>• Testing 15 E.coli+K1364014 clones
-
Date: 08/18/2014
+
<br>Date: 08/18/2014
-
Result: clones H, J, O, Q, U, V might have the right K1364014 construction
+
<br>Result: clones H, J, O, Q, U, V might have the right K1364014 construction
-
II. BBa_K1364014 in PsBs4s (K823022) and PsBs1C (K823023)
+
<br>2. BBa_K1364014 in PsBs4s (K823022) and PsBs1C (K823023)
-
• Digestion of K1364014, K823022 and K823023
+
<br>• Digestion of K1364014, K823022 and K823023
-
Date: 08/22/2014
+
<br>Date: 08/22/2014
-
Result: digestion of K13664014, K823022 and K823023 by EcoR1 and Pst1 were well performed.
+
<br>Result: digestion of K13664014, K823022 and K823023 by EcoR1 and Pst1 were well performed.
-
• Ligation of K1364014 with K823022 and K1364014 with K823023
+
<br>• Ligation of K1364014 with K823022 and K1364014 with K823023
-
Date: 08/22/2014
+
<br>Date: 08/22/2014
-
Result: We obtained 20µL ligation of K1364014 with K823022 and of K1364014 with K823023.
+
<br>Result: We obtained 20µL ligation of K1364014 with K823022 and of K1364014 with K823023.
-
• Transformation of ligations in E.coli
+
<br>• Transformation of ligations in E.coli
-
E. coli transformed by K1364014+K823022 spread on LB+Amp 100µg/mL agar plate
+
<br>E. coli transformed by K1364014+K823022 spread on LB+Amp 100µg/mL agar plate
-
E. coli transformed by K1364014+K823023 spread on LB+Amp 100µg/mL agar plate
+
<br>E. coli transformed by K1364014+K823023 spread on LB+Amp 100µg/mL agar plate
-
Date: 08/25/2014
+
<br>Date: 08/25/2014
-
• Testing E.coli+K1364014+K823022 and E.coli+K1364014+K823023 clones
+
<br>• Testing E.coli+K1364014+K823022 and E.coli+K1364014+K823023 clones
-
Date: 08/27/2014
+
<br>Date: 08/27/2014
-
3.  Transformation of K1364014+K823022 and K1364014+K823023 in B. subtilis
+
<br>3.  Transformation of K1364014+K823022 and K1364014+K823023 in B. subtilis
-
B. subtilis transformed by 10µL K1364014+K823022 spread on LB+Spec 75µg/mL agar plate
+
<br>B. subtilis transformed by 10µL K1364014+K823022 spread on LB+Spec 75µg/mL agar plate
-
B. subtilis transformed by 10µL K1364014+K823023 spread on LB+Cm 15µg/mL agar plate
+
<br>B. subtilis transformed by 10µL K1364014+K823023 spread on LB+Cm 15µg/mL agar plate
-
Date : 08/28/2014
+
<br>Date : 08/28/2014
-
Fungicides tests
+
<br>Fungicides tests
-
1. D4E1-GAFP1
+
<br>1. D4E1-GAFP1
-
• 08/12/2014 : Transformation in bacillus  Pveg-D4E1-GAFP1 on pSBBS4S  
+
<br>• 08/12/2014 : Transformation in bacillus  Pveg-D4E1-GAFP1 on pSBBS4S  
-
• 08/13/2014 :  integration threonine test + fungicide test  
+
<br>• 08/13/2014 :  integration threonine test + fungicide test  
-
2. D4E1
+
<br>2. D4E1
-
• 08/15/2014 : Cloning D4E1 into pSBBS1C + fungicide test
+
<br>• 08/15/2014 : Cloning D4E1 into pSBBS1C + fungicide test
-
• 08/19/2014 : D4E1 on pSB1C3 + fungicide test
+
<br>• 08/19/2014 : D4E1 on pSB1C3 + fungicide test
   
   
-
COMPLETER
 
</p>
</p>

Revision as of 16:31, 7 October 2014