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Team:Toulouse/Notebook/Project Monitoring
From 2014.igem.org
D4E1
1. Transformation of D4E1 (pEX-A2) into E. coli
Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
Date: 21/07/2014
2. Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli
Date: 22/07/2014
3. QIAprep Spin Miniprep Kit Using a Microcentrifuge
Date: 23/07/2014
4. Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
Date: 23/07/2014
5. Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
6. Cloning Pveg+D4E1 on psBbs4S
Date: 13/08/2014
7. Cloning Pveg+D4E1 on psBbs1C
Date: 20/08/2014
GAFP1
1. Transformation of GAFP1 (pEX-A2) into E. coli
Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
Date: 21/07/2014
• Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli
Date: 22/07/2014
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli
Date: 23/07/2014
• Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis
Date: 23/07/2014
2. Cloning GAFP1_pEX-A2 with BBa_K606013 (RFP_pSb1c3) into E. coli
• Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSb1c3)
Date: 23/07/2014
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: 25/07/2014
2. Spreading of coli cells transformed with pUC + Utah
Date: 28/07/2014
3. Liquid culture + Miniprep + Test of the miniprep
Date: 30/07/2014
4. Cloning 1: EcAMP + Pveg + RBS
• Digestion of EcAMP (INSERT) by XbaI and PstI
Date: 31/07/2014
• Digestion of Pveg + RBS (VECTOR)
Date: 31/07/2014
• Ligation and transformation
Date: 04/08/2014
• PCR test
Date: 05/08/2014
• Analytical digestion
Date: 05/08/2014
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
6. Striation on a petri dish to purify the clone
Purpose: to isolate a clone with vector+insert
Date: 07/08/2014
7. Miniprep of Pveg+SpoVG+EcAMP and analytic digestion
Date: 08/08/2014
8. Ligation of Pveg SpoVG EcAMP with double terminateur B0015
Transformation and liquid culture
Date: 11/08/2014
9. Miniprep of Pveg SpoVG EcAMP + analytic digestion
Date: 13/08/2014
10. Clonage K1364011 (EcAMP+Pveg +SpoVG + B0015) + K823022 (psBbs4S) : Digestion, ligation, transformation
Date: 19/08/2014
11. Clonage K1364011 + K823023 (psBbs1C) : Digestion, ligation, transformation
Date: 18/08/2014
12. Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test
Date: 21/08/2014
1. D4E1-GAFP1
• 12/08/2014 : Transformation in bacillus Pveg-D4E1-GAFP1 on PsBBS4S (+spectino)
• 13/08/2014 : integration threonine test + fungicide test
2. D4E1
• 15/08/2014 : Cloning D4E1 into psBbs1C + fungicide test
• 19/08/2014 : D4E1 on psb1C3 + fungicide test
1. Amplification of Binding Module (pEX-K4) into E.coli
• Transformation of Binding module (pEX-K4) into E. coli
Gene "Binding module" synthesized by Eurofins, resuspended in 20μL Tris 10mM
• Liquide culture of 2 clones : 1 et 2 (pEX-K4) transformed into E. coli
Date: 01/08/2014
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 2 clones of Binding Module (pEX-K4) into E. coli
Date: 04/08/2014
• Digestion of Binding Module (pEX-K4) with EcoRI and PstI
Date: 04/08/2014
2. Cloning Binding Module on pEX-K4 with pSB1C3 (BBa-K606013 without RFP) into E. coli
• Digestion Binding Module on pEX-K4 and BBa-K606013
Date: 23/08/2014
1. Transformation of chemotaxis (Puc-57) into E. coli
Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM
Date: 01/08/2014
2. Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into E. coli
Date: 04/08/2014
3. QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of chemotaxis (Puc57) into E. coli
Date: 05/08/2014
4. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested_pSb1c3 with EcoRI and PstI into E. coli
• Digestion BBa_K1364000 (chemotaxis_Puc57) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
Date: 07/08/2014
Result: Expected band after digestion for BBa_K1364000 : 2300bp
Problem: We can't distinguish the vector band (2500 bp)
• Gel extraction of BBa_K1364000
Date: 07/08/2014
• Ligation BBa_K1364000 and digested PsB1C3 with EcoRI and PstI
Date: 08/08/2014
• Transformation BBa_K1364000 in E.coli
Date: 08/08/2014
• Test of sensibility on Ampicillin
Date: 10/08/2014
Result: we can determine which colonies are sensible for ampicillin and know which baceria are carrying the chemotaxis gene
• PCR
Date: 11/08/2014
• Digestion BBa_K1364000 on PsB1C3 with EcoRI and PstI
Date: 11/08/2014
5. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on PsB1C3 with SpeI and PstI into E. coli
• Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI
Date: 08/08/2014
• Transformation BBa_1364004 in E.coli
Date: 8/08/2014
• Test of sensibility on Ampicilline
Date: 10/08/2014
• Digestion BBa_K1364004 on PsB1C3 with EcoRI and PstI
Date: 12/08/2014
6. Cloning chemotaxis BBa_K1364000 (chemotaxis_Puc57) with digested BBa_823003 (Pveg) on PsB1C3 with SpeI and PstI into E. coli
• Ligation BBa_K1364000 and digested BBa_823003 on PsB1C3 with SpeI and PstI
Date: 11/08/2014
• Transformation BBa_1364004 in E.coli
Date: 11/08/2014
• Test of sensibility on Ampicilline
Date: 14/08/2014
• Digestion BBa_K1364004 on PsB1C3 with EcoRI and PstI
Date: 18/08/2014
• Gel extraction of BBa_K1364000
Date: 19/08/2014
7. Cloning chemotaxis BBa_K1364004 with digested PsBS4S with EcorI and PstI into E. coli
• Ligation BBa_K1364004 digested EcorI and PstI and digested PsB1C3 with EcoRI and PstI
Date: 19/08/2014
• Transformation BBa_1364004 in PsBS4S in E.coli
Date: 19/08/2014
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