Difference between revisions of "Team:Aix-Marseille/Notebook"
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* Colony PCR of CsgA from the 01/08 using EconoTac ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR of CsgA from the 01/08 using EconoTac ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* PCR clean up of DesC(200ng/µL) and gel extraction of DesA (83ng/µL), DesB(53ng/µL), FliC <i>E. coli</i> ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * PCR clean up of DesC(200ng/µL) and gel extraction of DesA (83ng/µL), DesB(53ng/µL), FliC <i>E. coli</i> ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
| − | * SLIC of DesA, DesB, DesC, DesD and transformation of 10µL in 100µL | + | * SLIC of DesA, DesB, DesC, DesD and transformation of 10µL in 100µL competent Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). |
* Sequencage of Desulfovribrio clone 3 validated. | * Sequencage of Desulfovribrio clone 3 validated. | ||
* E/P digestion on pSB1C3, pSB1A3 and pSB1K3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.235_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * E/P digestion on pSB1C3, pSB1A3 and pSB1K3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.235_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
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[[File:T--Aix-Marseille--PCR-08-08.jpg|left|thumb|Colony PCR verification. The clones picked up on the plates are wroten on the right of the gel. the awaited size is 506bp for all clones.]] | [[File:T--Aix-Marseille--PCR-08-08.jpg|left|thumb|Colony PCR verification. The clones picked up on the plates are wroten on the right of the gel. the awaited size is 506bp for all clones.]] | ||
* Ligation of BBa_B0034+BBa_I0500 and Bba_K1951000 into pSB1K3 to create desA producer ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation of BBa_B0034+BBa_I0500 and Bba_K1951000 into pSB1K3 to create desA producer ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | * Transformation of the previous ligation in | + | * Transformation of the previous ligation in competent Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations protocol#2]). |
* Starters from DesC and Bba_K1951008 of the 08.04.2016. | * Starters from DesC and Bba_K1951008 of the 08.04.2016. | ||
* Resuspension of oligo using the IDT protocole. | * Resuspension of oligo using the IDT protocole. | ||
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* PCR using Q5 master with new FliC coli sequence (substitute Bbs1 site) Bba_K1951005 and Bba_K1951001, every SLIC have been validated by electrophoresis ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * PCR using Q5 master with new FliC coli sequence (substitute Bbs1 site) Bba_K1951005 and Bba_K1951001, every SLIC have been validated by electrophoresis ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* PCR clean up on the 2 previous SLIC ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * PCR clean up on the 2 previous SLIC ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
| − | * SLIC and Transformation of Bba_K1951005 and Bba_K1951001 in | + | * SLIC and Transformation of Bba_K1951005 and Bba_K1951001 in competent Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). |
* Starter of Bba_K1951004, Bba_K1951009 and Bba_K1951010 to make a protein production test. | * Starter of Bba_K1951004, Bba_K1951009 and Bba_K1951010 to make a protein production test. | ||
</div> | </div> | ||
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* Pcr clean up on the digestion from the 08.25.2016 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * Pcr clean up on the digestion from the 08.25.2016 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
* Ligation using 2A method of Bba_K1951002 or Bba_K1951003 and Bba_B0034 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation using 2A method of Bba_K1951002 or Bba_K1951003 and Bba_B0034 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | * Transformation of the previous ligation in | + | * Transformation of the previous ligation in competent Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
* Digestion of 500ng in 50µL total mix of Bba_K1951002 and Bba_K1951005 by XbaI and PstI ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion of 500ng in 50µL total mix of Bba_K1951002 and Bba_K1951005 by XbaI and PstI ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Ligation of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) overnight at 16°C ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) overnight at 16°C ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
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* Colony PCR of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Colony PCR of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) + BBa_I0500 (Prom) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) + BBa_I0500 (Prom) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | * Transformation of Bba_K1951004 (DesA with pSB1C3) in | + | * Transformation of Bba_K1951004 (DesA with pSB1C3) in competent Tg1 cells ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
* Ligation of Bba_K1951004 digested E/P in pSB1C3 at 16°C overnight ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation of Bba_K1951004 digested E/P in pSB1C3 at 16°C overnight ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
NB : we wanted to transform a cadA mutant from KEIO bank so we can't use this biobrick in pSB1K3. | NB : we wanted to transform a cadA mutant from KEIO bank so we can't use this biobrick in pSB1K3. | ||
* Starters from intermediate biobirck Bba_K1951001 (DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) and Bba_K1951000(DesA),Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) + BBa_I0500(Prom). | * Starters from intermediate biobirck Bba_K1951001 (DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) and Bba_K1951000(DesA),Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) + BBa_I0500(Prom). | ||
| − | * Made | + | * Made competent cells Tg1, culture from an over night starter have grown up from Abs(600nm)=0.05 until Abs(600nm)=0.457 at 37°C ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol#1]). |
</div> | </div> | ||
<div id="2016-09-01" class="evday"> | <div id="2016-09-01" class="evday"> | ||
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* Ligation of Bba_K1951004 (S/P) and Bba_K1951001 + RBS (X/P) in pSB1K3(E/P) at 16°C overnight ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation of Bba_K1951004 (S/P) and Bba_K1951001 + RBS (X/P) in pSB1K3(E/P) at 16°C overnight ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Ligation of Bba_K1951002 + RBS (E/S) and Bba_K1951003 + RBS (X/P) in pSB1K3(E/P) at 16°C overnight ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation of Bba_K1951002 + RBS (E/S) and Bba_K1951003 + RBS (X/P) in pSB1K3(E/P) at 16°C overnight ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | * Made | + | * Made competent cells of fliC mutant and cadA mutant from the Keio bank and test on different antibiotic petri dishes (only kanamycin dish was a positive control) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol#1]). |
* Retest of colony PCR on Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) + BBa_I0500(Prom) and nothing was good ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Retest of colony PCR on Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) + BBa_I0500(Prom) and nothing was good ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | * Transformation of the ligation Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) + BBa_I0500(Prom) from the ligation of the 08.26.2016 with the new | + | * Transformation of the ligation Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) + BBa_I0500(Prom) from the ligation of the 08.26.2016 with the new competent cells from 08.30.2016 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
| − | * Transformation of Bba_K1951008 in fliC mutant Keio | + | * Transformation of Bba_K1951008 in fliC mutant Keio competent made the 30th, spread on Kana/Cm petri dishes ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
* Made a calibration range for HPLC. | * Made a calibration range for HPLC. | ||
</div> | </div> | ||
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* Remade ligation of pSB1C3 (E/P) and Bba_K1951004 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Remade ligation of pSB1C3 (E/P) and Bba_K1951004 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | * Transformation of the previous ligation mix in | + | * Transformation of the previous ligation mix in competent Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
* Colony PCR on transformation of intermediate biobricks ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]).: | * Colony PCR on transformation of intermediate biobricks ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]).: | ||
- Bba_K1951004 + RBS (X/P) Bba_K1951001 in pSB1K3(E/P) at 16°C overnight | - Bba_K1951004 + RBS (X/P) Bba_K1951001 in pSB1K3(E/P) at 16°C overnight | ||
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=== Tuesday, September 6th === | === Tuesday, September 6th === | ||
[[File:T--Aix-Marseille--PCR-06-09.jpg|left|thumb|PCR colony verification. The holes 2 to 7 are the clone 12 of the plate and 11 to 16 are the clone 13 of the plate. The holes 9 and 17 are the RPF (control).]] | [[File:T--Aix-Marseille--PCR-06-09.jpg|left|thumb|PCR colony verification. The holes 2 to 7 are the clone 12 of the plate and 11 to 16 are the clone 13 of the plate. The holes 9 and 17 are the RPF (control).]] | ||
| − | * Transformation of cadA mutant | + | * Transformation of cadA mutant competent strain by Bba_K1951004 in pSB1C3, spread on Kana/Cm petri dishes ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
* Colony PCR Bba_K1951004 in pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR Bba_K1951004 in pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Starters from Bba_K1951004 in pSB1C3. | * Starters from Bba_K1951004 in pSB1C3. | ||
* 1 step of the transduction using phage p1 from fliC mutant Keio bank. Conserved at 4°C with 20µL chlorophorme | * 1 step of the transduction using phage p1 from fliC mutant Keio bank. Conserved at 4°C with 20µL chlorophorme | ||
* PCR with Q5 master mix using oligo of diriged mutagenesis for the insertion of Bbs1 site in the 214-215 and 238-239 sites of BBa_K1951008. Digest 3h with addition of 1µL DpNI in the PCR tube ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * PCR with Q5 master mix using oligo of diriged mutagenesis for the insertion of Bbs1 site in the 214-215 and 238-239 sites of BBa_K1951008. Digest 3h with addition of 1µL DpNI in the PCR tube ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | * Transformation of the previous PCR in | + | * Transformation of the previous PCR in competent Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
* Starters of the PCR from 09.02.2016. | * Starters of the PCR from 09.02.2016. | ||
* Remade PCR on Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) + BBa_I0500(Prom) but still no result ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Remade PCR on Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) with Bba_B0034 (rbs) + BBa_I0500(Prom) but still no result ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
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* Digestion of intermediate biobrick BBa_K1951004+ RBS BBa_K1951001 by EcoRI and SpeI and RBS BBa_K1951001+ RBS BBa_K1951002 by SpeI and PstI ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion of intermediate biobrick BBa_K1951004+ RBS BBa_K1951001 by EcoRI and SpeI and RBS BBa_K1951001+ RBS BBa_K1951002 by SpeI and PstI ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Ligation of the 2 previous digestions in pSB1C3 to make the final BBa_K1951011 (E/P) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation of the 2 previous digestions in pSB1C3 to make the final BBa_K1951011 (E/P) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | * Transformation of the previous ligation in | + | * Transformation of the previous ligation in competent Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
</div> | </div> | ||
<div id="2016-09-09" class="evday"> | <div id="2016-09-09" class="evday"> | ||
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=== Sunday, September 11st === | === Sunday, September 11st === | ||
| − | * Made | + | * Made competent of the fliC mutant W3110 strain from 5 differents colonies from 4 colonies which swam well ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol#1]). |
| − | * Transformation of the previous | + | * Transformation of the previous competent with BBa_K1951008 and BBa_K1951009 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
* Transformation of cadA mutant Keio strain with BBa_K1951011 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of cadA mutant Keio strain with BBa_K1951011 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
</div> | </div> | ||
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=== Monday, September 12rd === | === Monday, September 12rd === | ||
| − | * Transformation of the fliC mutant W3110 | + | * Transformation of the fliC mutant W3110 competent with BBa_K1951008 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). |
</div> | </div> | ||
<div id="2016-09-13" class="evday"> | <div id="2016-09-13" class="evday"> | ||
