Team:Toulouse/Notebook/Project Monitoring
From 2014.igem.org
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<br/>Date: 07/23/2014 | <br/>Date: 07/23/2014 | ||
<br/>Results : band at 860 bp for BBa_K606013 | <br/>Results : band at 860 bp for BBa_K606013 | ||
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<p><br/><FONT SIZE= 4%>12. Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test | <p><br/><FONT SIZE= 4%>12. Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test | ||
</FONT><br/>Date: 08/21/2014 | </FONT><br/>Date: 08/21/2014 | ||
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+ | <p> | ||
+ | <br/><B> Assembling the fungicides module </B> | ||
+ | <p> | ||
+ | <br/>-Cloning D4E1 + GAFP1 | ||
+ | <br/>Date: 08/05/2014 | ||
+ | <br/>-Cloning Pveg + D4E1-GAFP1 | ||
+ | <br/>Date: 08/08/2014 | ||
+ | <br/>-Cloning Pveg-D4E1-GAFP1 on pSBBS4S | ||
+ | <br/>Date : 08/12/2014 | ||
+ | <br/>Threonine test on 10 colonies (streak on plates with and without thr) | ||
+ | <br/>Date : 08/13/2014 | ||
+ | <br/>D4E1-GAFP1-EcAMP : BBa_K1364014 | ||
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+ | <p><br/><FONT SIZE= 4%>1. Digestion of K1364010 and K1364012</FONT> | ||
+ | <br/>• Digestion K13664010 by Spe1 and Pst1, and digestion of K1364012 by XbaI and Pst1. | ||
+ | <br/>• Kit clean up to remove enzymes from digestion | ||
+ | <br/>Date: 07/11/2014 | ||
+ | <br/>• Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli | ||
+ | <br/>Date: 07/22/2014 | ||
+ | <br/>• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli | ||
+ | <br/>Processing details: Elution Buffer at 50-55°C | ||
+ | <br/>Date: 23/07/2014 | ||
+ | <br/>• Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis | ||
<p> | <p> |
Revision as of 08:14, 11 September 2014
D4E1
1. Transformation of D4E1 (pEX-A2) into E. coli
Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
Date: 07/21/2014
2. Culture of 4 clones: A, B, C, D of D4E1 (pEX-A2) transformed into E. coli
Date: 07/22/2014
3. QIAprep Spin Miniprep Kit Using a Microcentrifuge
Date: 07/23/2014
4. Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
Date: 07/23/2014
5. Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
6. Cloning Pveg+D4E1 on psBbs4S
Date: 08/13/2014
7. Cloning Pveg+D4E1 on psBbs1C
Date: 08/20/2014
8. Tests
- Cloning D4E1 into Bacillus pSBBS4S (spectino 75µg/ml) , and fungicide test
Date: 08/15/2014
Results :10 clones tested, each colony was tested on a PDA plate + fungi spread as a cross => fail
New cloning on pSBBS4S => threonine test is OK for 2 clones
GAFP1
1. Transformation of GAFP1 BBa_K78002(pEX-A2) into E. coli
Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
Date: 07/21/2014
• Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli
Date: 07/22/2014
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli
Date: 07/23/2014
• Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis
Date: 07/23/2014
2. Cloning GAFP1_pEX-A2 with BBa_K606013 (RFP_pSB1C3) into E. coli
• Digestion GAFP1_pEX-A2 and BBa_K606013 (RFP_pSb1c3)
Date: 07/23/2014
Results : band at 860 bp for BBa_K606013
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
Date: 07/28/2014
3. Liquid culture + Miniprep + Test of the miniprep
Date: 07/30/2014
4. Cloning 1: EcAMP + Pveg + RBS
• Digestion of EcAMP (INSERT) by XbaI and PstI
Date: 07/31/2014
• Digestion of Pveg + RBS (VECTOR)
Date: 07/31/2014
• Ligation and transformation
Date: 08/04/2014
• PCR test
Date: 08/05/2014
• Analytical digestion
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: 08/06/2014
• Digestion of EcAMP (INSERT) by XbaI and PstI
• Digestion of Pveg + RBS (VECTOR)
• Heat inactivation of the enzymes
• Ligation and transformation
• PCR test
6. Striation on a petri dish to purify the clone
Purpose: to isolate a clone with vector+insert
Date: 08/07/2014
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: 08/11/2014
9. Miniprep of Pveg SpoVG EcAMP + analytic digestion
Date: 08/13/2014
10. Clonage K1364011 (EcAMP+Pveg +SpoVG + B0015) + K823022 (psBbs4S) : Digestion, ligation, transformation
Date: 08/19/2014
11. Clonage K1364011 + K823023 (psBbs1C) : Digestion, ligation, transformation
Date: 08/18/2014
12. Verification of the insertion of K1364011 + K823022 (pSBBS4S) into the subtilis genome by threonine test
Date: 08/21/2014
Assembling the fungicides module
-Cloning D4E1 + GAFP1
Date: 08/05/2014
-Cloning Pveg + D4E1-GAFP1
Date: 08/08/2014
-Cloning Pveg-D4E1-GAFP1 on pSBBS4S
Date : 08/12/2014
Threonine test on 10 colonies (streak on plates with and without thr)
Date : 08/13/2014
D4E1-GAFP1-EcAMP : BBa_K1364014
1. Digestion of K1364010 and K1364012
• Digestion K13664010 by Spe1 and Pst1, and digestion of K1364012 by XbaI and Pst1.
• Kit clean up to remove enzymes from digestion
Date: 07/11/2014
• Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli
Date: 07/22/2014
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of GAFP1 (pEX-A2) into E. coli
Processing details: Elution Buffer at 50-55°C
Date: 23/07/2014
• Digestion of GAFP1 (pEX-A2) with EcoRI and PstI - Inactivation EcoRI - PCR kit Clean up to remove PstI - Gel Electrophoresis
1. D4E1-GAFP1
• 08/12/2014 : Transformation in bacillus Pveg-D4E1-GAFP1 on PsBBS4S (+spectino)
• 08/13/2014 : integration threonine test + fungicide test
2. D4E1
• 08/15/2014 : Cloning D4E1 into psBbs1C + fungicide test
• 08/19/2014 : 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: 08/01/2014
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 2 clones of Binding Module (pEX-K4) into E. coli
Date: 08/04/2014
• Digestion of Binding Module (pEX-K4) with EcoRI and PstI
Date: 08/04/2014
Results: 3 bands are obtained at 1500 bp (vector), 1300 bp (binding gene) and 1000bp (vector with pUC replication origin)
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: 08/04/2014
- BBa_K606013 digested by EcoRI and PstI
- Digestion of 2 Binding Module Miniprep with EcoRI and PstI
- PCR kit Clean up
- Gel Electrophoresis
Results:
BBa_K606013 : 860 bp band for RFP and 2100 bp for vector pSB1C3
Binding Module : 1400 bp for Binding Module and 1500+1000bp for vector pEX_K4
We decide to do a ligation between pSB1C3 and Binding Module without gel cut and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) in the aim to clean enzymes.
• Ligation Binding Module on pSB1C3
• Transformation of Binding Module on pSB1C3 into E. coli
Dated : 08/04/2014
3. Cloning Binding Module on pEX-K4 with pVeg on pSB1C3 (BBa-K823003) into E. coli
• Digestion Binding Module on pEX-K4 and BBa-K823003 by Xba and PstI
Results:
Binding Module = 1400 bp for Binding Module and 1500+1000bp for vector pEX_K4
We decide to do a ligation between pVeg and Binding Module without gel cut and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) in the aim to clean enzymes.
• Ligation Binding Module on pVeg
• Transformation of Binding Module on pVeg into E. coli
Dated : 08/04/2014
Results: Many good clones (check on 08/05/2014)
1. Transformation of chemotaxis gene BBa_K1364000(Puc-57) into E. coli
Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM
Date: 08/01/2014
2. Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into E. coli
Date: 08/04/2014
3. QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of chemotaxis (Puc57) into E. coli
Date: 08/05/2014
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: 08/07/2014
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: 08/07/2014
• 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: 08/10/2014
Result: we can determine which colonies are sensible for ampicillin and know which baceria are carrying the chemotaxis gene
• PCR
Date: 08/11/2014
• Digestion BBa_K1364000 on PsB1C3 with EcoRI and PstI
Date: 08/11/2014
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: 08/08/2014
• Test of sensibility on Ampicilline
Date: 08/10/2014
• Digestion BBa_K1364004 on PsB1C3 with EcoRI and PstI
Date: 08/12/2014
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: 08/11/2014
• Transformation BBa_1364004 in E.coli
Date: 08/11/2014
• Test of sensibility on Ampicilline
Date: 08/14/2014
• Digestion BBa_K1364004 on PsB1C3 with EcoRI and PstI
Date: 08/18/2014
• Gel extraction of BBa_K1364000
Date: 08/19/2014
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: 08/19/2014
• Transformation BBa_1364004 in PsBS4S in E.coli
Date: 08/19/2014