Team:Toulouse/Notebook/project-monitoring

Binding module

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
Result: We obtained distinct colonies on plates LB + Kanamycin (50 µg/mL)

Liquid culture of 2 clones : 1 et 2 (pEX-K4) transformed into E. coli

Date: 01/08/2014

Miniprep with QIAprep Spin Miniprep Kit: 2 clones of Binding Module (pEX-K4) into E. coli

Date: 04/08/2014
Result: Binding Module (pEX-K4) obtained

Digestion of Binding Module (pEX-K4) with EcoRI and PstI

Date: 04/08/2014
Result:
- 3 bands : 1500bp (vector with Kanamycin resistance), 1300bp (Module Binding) and 1000p bp (vector with pUC Ori)
- The two Binding Module clones are ok

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
Expected bands after digestion for:
- BBa_K606013 : 860 bp for RFP and 2100 bp for vector pSB1C3
- Binding Module : 1400 bp for Binding Module and 1500bp+1000bp for vector pEX_K4
We decide to do a ligation between pSB1C3 and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.

Ligation Binding Module on pSB1C3

Date: 04/08/2014

Transformation of Binding Module on pSB1C3 into E. coli

Date: 04/08/2014
Transformation with 10 µL of the ligation mix and plate on Chloremphenicol LA plate
Result : many wrong clones

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

Date: 04/08/2014
BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI
Gel Electrophoresis
Result:
- Binding Module : 1400 bp for Binding Module and 1500bp+1000bp for vector pEX_K47
We decide to do a ligation between pVeg and Binding Module without gel cutting and purification but after a PCR clean-up (Thermo scientific, GeneJET PCR purifiction kit #K0701) to remove the enzymes.

Ligation Binding Module on pVeg with pSB1C3

Date: 04/08/2014
BBa_K823003 digested by XbaI and PstI and Binding Module digested by SpeIand PstI and ligation

Transformation of Binding Module on pVeg into E. coli

Date: 04/08/2014
Result: Many good clones (check on 06/08/2014)

4. Cloning Binding Module with Pveg (BBa-K1364005) on pSBBS4S (BBa-K823022) into E. coli

Digestion Binding Module with Pveg (BBa-K1364005) and pSBBS4S (BBa-K823022)

Date: 07/08/2014
BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRIand PstIGel Electrophoresis
Result:
- Binding Module with Pveg 1600 bp for Binding Module and 2100bp for vector pSB1C3

Ligation Binding Module with Pveg on pSBbs4S

Date: 07/08/2014
BBa_K823022 and Binding Module with Pveg (BBa_K1364005) digested by EcoRIand PstI
Ligation
Result: Ligation between Binding Module with Pveg on pSBBS4S

Transformation of Binding Module with Pveg on pSBBS4S into E. coli

Date: 04/08/2014
Binding Module with Pveg on pSBbs4S
Plate on Ampicillin LA plate
Result: Many good clones (check on 13/08/2014)

Transformation of Binding Module with Pveg on pSBBS4S into B. subtilis

Date: 04/08/2014
After 5 hours of incubation and the linearization of 6µl of DNA with 1µl of ScaI, we make the transformation. Plate on Spectinomycin LA plate.
Result : One good clone : PCR (check on 09/09/2014) and threonine test (check on 09/09/2014)

5. Binding Test

See here

Chemotaxis

1. Transformation of chemotaxis (Puc-57) into E. coli

Gene chemotaxis synthesized by Eurofins, resuspended in 20μL Tris 10mM
Date: 01/08/2014
Result: We obtained distinct colonies on LA + Ampicillin (100 µg/mL) plates

Culture of 4 clones: A, B, C, D of chemotaxis (Puc57) transformed into E. coli

Date: 04/08/2014

Miniprep with QIAprep Spin Miniprep Kit Using a Microcentrifuge: 4 clones of chemotaxis (Puc57) into E. coli

Date: 05/08/2014
Result: 4*50µL of chemotaxis (Puc57) obtained

2. 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 - PCR kit Clean up

Date: 07/08/2014
Result: Expected band after digestion for BBa_K1364000: 2300 bp
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
Result: We obtained distinct colonies on LA + Cm (15 µg/mL) plates and resuspended 15 colonies in LB+Cm (15 µg/mL)

Test of sensibility on Ampicillin

Date: 10/08/2014
Result: we can determine which colonies are sensible for ampicillin and know which bacterium is carrying the chemotaxis gene.

PCR

Date: 11/08/2014

Digestion BBa_K1364000 on pSB1C3 with EcoRI and PstI

Date: 11/08/2014
Result: There is one colony which presents the right construction.

3. 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 Ampicillin
Date: 10/08/2014
Result : We can determine which colony is sensible for ampicillin and know which bacterium is carrying the chemotaxis gene. There is one colony which resists on ampicillin.
• Digestion BBa_K1364004 on pSBC3 with EcoRI and PstI
Date: 12/08/2014
Result: We did not see any colony with chemotaxis insert.
4. 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 D
ate: 11/08/2014
• Transformation BBa_1364004 in E.coli
Date: 11/08/2014
• Test of sensibility on Ampicillin
Date: 14/08/2014
Result: we obtained 4 colonies sensible at Ampicilline
• Digestion BBa_K1364004 on PsB1C3 with EcoRI and PstI
Date: 18/08/2014
• Gel extraction of BBa_K1364000
Date: 19/08/2014
5. Cloning chemotaxis BBa_K1364004 with digested pSBBS4S 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 pSBBS4S in E.coli
Date: 19/08/2014
Result : We obtained one colony and resuspended it in LB+ Amp

Fungicides

D4E1
1. Amplification of synthetic gene (D4E1 on pEX-A2)
• Transformation of D4E1 (pEX-A2) into E. coli
Gene D4E1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
Concentration of D4E1 : 115ng/µL
Date: 07/21//2014
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
Date: 07/22/2014
Result : Culture of 4 clones ok
• QIAprep Spin Miniprep Kit Using a Microcentrifuge : 4 clones of D4E1 (pEX-A2) into E. coli
Buffer EB at 50-55°C
Date: 07/23/2014
• Digestion of D4E1 (pEX-A2) with EcoRI and PstI - Stop EcoRI - PCR kit Clean up
Date: 07/23/2014
Result : 4*20µL D4E1 digested with EcoRI and PstI  all the clones seem to have the right D4E1 gene.
2. Cloning D4E1 in pSB1C3
• Digestion of D4E1 on pEX-A2 and pSB1C3
Date: 07/23/2014
• Ligation of D4E1 in pSB1C3
Date: 07/23/2014
• Transformation in E.coli
Date : 07/23/2014
• 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
Finished by: Emeline and Diane
Dated: 07/24/2014
Result: Culture of 4 clones ok
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
Date: 07/25/2014
• Digestion of D4E1 (pSB1C3) A, B, C, D, E, F with EcoRI and PstI - PCR kit Clean up + electrophoresis
Date: 07/25/2014
Result: clones C, D have the expected construction
• PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
Date: 07/25/2014
Result: clones C, D have the expected construction, and placed in cryopreservation.
3. Cloning Pveg+D4E1 on Pveg plasmid (pSB1C3)
• Digestion of D4E1 on pEX-A2 and K823003 (Pveg on pSB1C3)
Finished by: Emeline and Diane
Dated: 07/24/2014
Result: 20µL digestion of D4E1 on pEX-A2 and of K823003
• Ligation of D4E1 in K823003 (Pveg on pSB1C3)
Date: 07/24/2014
Result: 20µL ligation of D4E1 in K823003
• Transformation of ligation products in E.coli
Date : 07/24/2014
Result: E.coli transformed by D4E1+K823003
• Culture of 6 clones: A, B, C, D, E, F of transformed E. coli
Date: 07/26/2014
Result: Culture of 4 clones ok
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
Date: 07/28/2014
• PCR of D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
Dated : 07/28/2014
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
Date: 28/07/2014
Result : clones C, D have the expected construction and are placed in cryopreservation.
4. Cloning Pveg+D4E1 on pSBBS4S (K823022)
Date: 08/13/2014
5. Cloning Pveg + D4E1 on pSBBS1C lacZ (23)
GAFP1
1. Amplification of synthetic gene (GAFP1 on pEX-A2)
• Transformation of GAFP1 (pEX-A2) into E.coli
Gene GAFP1 synthesized by Eurofins, resuspended in 20μL Tris 10mM
Concentration of GAFP1 : 145ng/µL
Date : 07/21/2014
Result: We obtained distinct colonies on plates LB + Ampicillin (100 µg/mL)
• Culture of 4 clones: A, B, C, D of GAFP1 (pEX-A2) transformed into E. coli
Date: 07/22/2014
Result: Culture of 4 clones ok
• 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: 07/23/201
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)
• Digestion of GAFP1 on pEX-A2 and B0015 (terminator on pSB1C3)
Date : 07/24/2014
• Ligation of GAFP1 in B0015 (terminator on pSB1C3)
Date: 07/24/2014
• Transformation of ligation products in E.coli
Date: 07/24/2014
• Culture of 6 clones: A, B, C, D, E, F transformed in E. coli
Date: 07/26/2014
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
Date : 07/28/2014
• PCR of GAFP1+B0015 = K1364007 (pSB1C3) A, B, C, D, E, F + electrophoresis
Date: 07/28/2014
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
Date: 07/28/2014
Result : clones B, C, D, E, F have the expected construction.
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
Date: 07/29/2014
• Gel extraction of K1364007 (extraction of GAFP1+ter gene)
Date: 07/29/2014
• Ligation of GAFP1+ter in K823003 (Pveg on pSB1C3)
Date : 07/29/2014
Result : 20µL ligation of GAFP1+ter in K823003
• Transformation of ligation products in E.coli
Date: 07/29/2014
• Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli
Date: 07/30/2014
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
Date : 07/31/2014
• PCR of GAFP1+B0015 + K823003 = K1364008 (pSB1C3) A, B, C, D, E, F, G, H + electrophoresis
Date: 31/07/2014
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
Date: 31/07/2014
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)
• Digestion of Pveg+GAFP1+ ter B0015 (K1364008) on pSB1C3 and PsBBs4S (K823022)
Date: 08/01/2014
• Ligation of K134008 (Pveg+GAFP1+ter fragment) and K823022 (PsBBs4S)
Date: 08/01/2014
• Transformation of ligation products in E.coli
Date : 08/01/2014
• Culture of 8 clones: A, B, C, D, E, F, G, H of transformed E. coli
Date: 08/02/2014
• QIAprep Spin Miniprep Kit Using a Microcentrifuge
Date: 08/04/2014
• Digestion of clones A, B, C, D, E, G, H of K1364008+K823022 with EcoRI and PstI + electrophoresis
Date: 08/04/2014
Result: clones A, E, F have the right construction
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: 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
Assembling the fungicides module
D4E1 + GAFP1
1. Cloning GAFP1+D4E1 on pSB1C3: BBa_K1364012
• Digestion of D4E1 on pEX-A2 and GAFP1 on pSB1C3
Date: 07/28/2014
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
Date: 07/28/2014
• Transformation of ligation of GAFP1 and D4E1 on pSB1C3 into E. coli
Date: 07/28/2014
• PCR of GAFP1+D4E1 (pSB1C3) A, B, C, D, E, F + electrophoresis
Date: 07/29/2014
Result: clones A, C, F, G seem to have the expected construction.
• Digestion of GAFP1+D4E1 (pSB1C3) A, C, F, G + electrophoresis
Date: 07/30/2014
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
3. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS4S (K823022)
4. Cloning Pveg+GAFP1+D4E1 (BBa_K1364013) on pSBBS1C lacZ (K823023)
5. Fungicides tests
Cloning D4E1-GAFP1-EcAMP: BBa_K1364014
1. Construction of BBa_K1364014 in PsB1C3, in E. coli
• Digestion of K1364010 and K1364012
Digestion of K13664010 by Spe1 and Pst1, and digestion of K1364012 by XbaI and Pst1.
Date: 08/11/2014
Result : We obtained digested fragments of K1364012 and ~500 bp fragment of K1364010
• Ligation of ~500 bp fragment from K1364010 and K1364012
Date: 08/11/2014
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
Date: 08/12/2014
• Testing 8 E.coli+K1364014 clones
Date: 08/14/2014
• Testing 15 E.coli+K1364014 clones
Date: 08/18/2014
Result: clones H, J, O, Q, U, V might have the right K1364014 construction
2. BBa_K1364014 in PsBs4s (K823022) and PsBs1C (K823023)
• Digestion of K1364014, K823022 and K823023
Date: 08/22/2014
Result: digestion of K13664014, K823022 and K823023 by EcoR1 and Pst1 were well performed.
• Ligation of K1364014 with K823022 and K1364014 with K823023
Date: 08/22/2014
Result: We obtained 20µL ligation of K1364014 with K823022 and of K1364014 with K823023.
• Transformation of ligations in E.coli
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
Date: 08/25/2014
• Testing E.coli+K1364014+K823022 and E.coli+K1364014+K823023 clones
Date: 08/27/2014
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
B. subtilis transformed by 10µL K1364014+K823023 spread on LB+Cm 15µg/mL agar plate
Date : 08/28/2014
Fungicides tests
1. D4E1-GAFP1
• 08/12/2014 : Transformation in bacillus Pveg-D4E1-GAFP1 on pSBBS4S
• 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