Team:Toulouse/Notebook/Project Monitoring

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

iGEM Toulouse INSA Toulouse 135, Avenue de Rangueil 31400 TOULOUSE e-mail: igemtoulouse2014@gmail.com