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 : 1500 bp (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 Result : 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. BindingTest 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 Date: 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 A COMPLETER 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) COMPLETER ! 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 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 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 II. 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 COMPLETER