Difference between revisions of "Team:Aix-Marseille/Notebook"
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* Preparation of heat competent DH5α cells ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol #1]). | * Preparation of heat competent DH5α cells ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol #1]). | ||
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</div> | </div> | ||
<div id="2016-06-14" class="evday"> | <div id="2016-06-14" class="evday"> | ||
| Line 167: | Line 165: | ||
* Transformation of DH5α cells with test plasmid ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of DH5α cells with test plasmid ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
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</div> | </div> | ||
<div id="2016-06-15" class="evday"> | <div id="2016-06-15" class="evday"> | ||
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* Preparation of heat competent Tg1 cells ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol#1]). | * Preparation of heat competent Tg1 cells ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol#1]). | ||
* Transformation of Tg1 cells with test plasmid ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of Tg1 cells with test plasmid ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
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</div> | </div> | ||
<div id="2016-06-16" class="evday"> | <div id="2016-06-16" class="evday"> | ||
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* Transformation of DH5α cells with pSB1C3 + RFP ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of DH5α cells with pSB1C3 + RFP ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
* Transformation with K880005, K823017, I0500, K1404006, B0034 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation with K880005, K823017, I0500, K1404006, B0034 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
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</div> | </div> | ||
<div id="2016-06-17" class="evday"> | <div id="2016-06-17" class="evday"> | ||
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* Plasmids purification K880005, K823017, I0500, K1404006, B0034 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmids purification K880005, K823017, I0500, K1404006, B0034 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
* Plasmids purification pSB1C3 + RFP ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmids purification pSB1C3 + RFP ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
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</div> | </div> | ||
<div id="2016-06-20" class="evday"> | <div id="2016-06-20" class="evday"> | ||
| Line 204: | Line 195: | ||
* Biobricks plasmids purifications gel migration ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Biobricks plasmids purifications gel migration ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
* Transformation Tg1 with RFP+pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]) | * Transformation Tg1 with RFP+pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]) | ||
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</div> | </div> | ||
<div id="2016-06-21" class="evday"> | <div id="2016-06-21" class="evday"> | ||
| Line 214: | Line 203: | ||
* E. Coli and Desulfo FliC (E/P digested) ligation in linearized pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * E. Coli and Desulfo FliC (E/P digested) ligation in linearized pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Transformation of pSB1C3 RFP in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of pSB1C3 RFP in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
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</div> | </div> | ||
<div id="2016-06-22" class="evday"> | <div id="2016-06-22" class="evday"> | ||
| Line 229: | Line 216: | ||
* Plasmid purification B0034 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmid purification B0034 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
* Transformation in DH5α of interlab studies plasmids ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation in DH5α of interlab studies plasmids ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
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</div> | </div> | ||
<div id="2016-06-23" class="evday"> | <div id="2016-06-23" class="evday"> | ||
| Line 244: | Line 229: | ||
[[File:T--Aix-Marseille--PCR-23-06.jpg|left|thumb|<u>Electrophoresis revelation</u> of E/P digestion on pSB1C3. Awaited size of 2070 bp. The two samples are the same digestion and we did a gel purification on the awaited band.]] | [[File:T--Aix-Marseille--PCR-23-06.jpg|left|thumb|<u>Electrophoresis revelation</u> of E/P digestion on pSB1C3. Awaited size of 2070 bp. The two samples are the same digestion and we did a gel purification on the awaited band.]] | ||
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</div> | </div> | ||
<div id="2016-06-24" class="evday"> | <div id="2016-06-24" class="evday"> | ||
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[[File:T--Aix-Marseille--PCR-24-06.jpg|left|thumb|Verification of the plasmid purifications on pSB1C3 pSB1CA3 pSB1CK3 pSB1CT3 (the name are wroten on the top of the gel) the awaited sizes are 2070bp.]] | [[File:T--Aix-Marseille--PCR-24-06.jpg|left|thumb|Verification of the plasmid purifications on pSB1C3 pSB1CA3 pSB1CK3 pSB1CT3 (the name are wroten on the top of the gel) the awaited sizes are 2070bp.]] | ||
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</div> | </div> | ||
<div id="2016-06-27" class="evday"> | <div id="2016-06-27" class="evday"> | ||
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* Transformations failed ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformations failed ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
* Transformation pSB1T3 plasmid purification, pSB1K3 plasmid purification, control ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.234_:_Plasmid_DNA_purification Protocol#4] and[https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation pSB1T3 plasmid purification, pSB1K3 plasmid purification, control ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.234_:_Plasmid_DNA_purification Protocol#4] and[https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
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</div> | </div> | ||
<div id="2016-06-28" class="evday"> | <div id="2016-06-28" class="evday"> | ||
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* PCR of IDT sequences (fliC and DesA to D) and gel check ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * PCR of IDT sequences (fliC and DesA to D) and gel check ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Gel extract pSB1CK3, pSB1CC3, pSB1CT3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * Gel extract pSB1CK3, pSB1CC3, pSB1CT3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
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</div> | </div> | ||
<div id="2016-06-29" class="evday"> | <div id="2016-06-29" class="evday"> | ||
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* Redone PCR of IDT sequences (fliC and DesA to D) (different Tm) and gel extract ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3] and [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * Redone PCR of IDT sequences (fliC and DesA to D) (different Tm) and gel extract ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3] and [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
* Transformation of fliCs from remaining ligation in Tg1 Sandra ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of fliCs from remaining ligation in Tg1 Sandra ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
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</div> | </div> | ||
<div id="2016-06-30" class="evday"> | <div id="2016-06-30" class="evday"> | ||
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* Migration FliC coli, desulfo, DesB, DesC ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Migration FliC coli, desulfo, DesB, DesC ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
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</div> | </div> | ||
<div id="2016-07-01" class="evday"> | <div id="2016-07-01" class="evday"> | ||
| Line 307: | Line 284: | ||
* Digestion PCR products and vector pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.235_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion PCR products and vector pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.235_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* PCR clean up on digested pSB1C3, fliC coli desulfo, DesB and DesC, migration and transformation in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * PCR clean up on digested pSB1C3, fliC coli desulfo, DesB and DesC, migration and transformation in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
| − | |||
[[File:T--Aix-Marseille--PCR-29-06.jpg|left|thumb|Verification of the plasmid purifications on pSB1C3.]] | [[File:T--Aix-Marseille--PCR-29-06.jpg|left|thumb|Verification of the plasmid purifications on pSB1C3.]] | ||
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</div> | </div> | ||
<div id="2016-07-04" class="evday"> | <div id="2016-07-04" class="evday"> | ||
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* Isolated white colonies on dishes | * Isolated white colonies on dishes | ||
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</div> | </div> | ||
<div id="2016-07-05" class="evday"> | <div id="2016-07-05" class="evday"> | ||
| Line 325: | Line 298: | ||
* Reisolated colonies from 04/07 | * Reisolated colonies from 04/07 | ||
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</div> | </div> | ||
<div id="2016-07-06" class="evday"> | <div id="2016-07-06" class="evday"> | ||
| Line 333: | Line 304: | ||
* Colony PCR with FLiC E.Coli Desulfo DesB DesC ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR with FLiC E.Coli Desulfo DesB DesC ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | |||
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</div> | </div> | ||
<div id="2016-07-07" class="evday"> | <div id="2016-07-07" class="evday"> | ||
| Line 343: | Line 312: | ||
* PCR on A2 A4 B1 clones from 6/7 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * PCR on A2 A4 B1 clones from 6/7 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Digestion E/P of A5 B4 C3 C2 minipreps and pSB1A3, pSB1C3, pSB1K3, pSB1T3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion E/P of A5 B4 C3 C2 minipreps and pSB1A3, pSB1C3, pSB1K3, pSB1T3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | |||
[[File:T--Aix-Marseille--PCR-07-07.jpg|left|thumb|Colony PCR from 06/07. For the two first holes, the awaited size is 1547 bp, for the two last, this size is 994bp]] | [[File:T--Aix-Marseille--PCR-07-07.jpg|left|thumb|Colony PCR from 06/07. For the two first holes, the awaited size is 1547 bp, for the two last, this size is 994bp]] | ||
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</div> | </div> | ||
<div id="2016-07-08" class="evday"> | <div id="2016-07-08" class="evday"> | ||
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* PCR clean up of vectors ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * PCR clean up of vectors ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
* PCR SLIC FliC coli/desulfo, DesA, B, C, D, csgA -> failed ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * PCR SLIC FliC coli/desulfo, DesA, B, C, D, csgA -> failed ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-11" class="evday"> | <div id="2016-07-11" class="evday"> | ||
| Line 369: | Line 334: | ||
* Transformation Tg1 with fliC coli desulfo, desA, desB in pSB1A3 from SLIC ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation Tg1 with fliC coli desulfo, desA, desB in pSB1A3 from SLIC ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
* PCR clean up desA, desB, fliC coli desulfo ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * PCR clean up desA, desB, fliC coli desulfo ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-12" class="evday"> | <div id="2016-07-12" class="evday"> | ||
| Line 382: | Line 345: | ||
* Transformation SLIC mix in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation SLIC mix in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
* Transformation constructions #7 (kanamycin) 9 and 55 (chloramphenicol) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation constructions #7 (kanamycin) 9 and 55 (chloramphenicol) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-13" class="evday"> | <div id="2016-07-13" class="evday"> | ||
| Line 393: | Line 354: | ||
* Colony PCR #1 2 3 50 51 53 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR #1 2 3 50 51 53 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Plasmid purification pSB1 A/C/K/T 3, gel migration ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmid purification pSB1 A/C/K/T 3, gel migration ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-18" class="evday"> | <div id="2016-07-18" class="evday"> | ||
| Line 406: | Line 365: | ||
* Digestion E/P and PCR clean up empty vectors A3 C3 K3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion E/P and PCR clean up empty vectors A3 C3 K3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* PCR clean up on A3, C3 and K3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * PCR clean up on A3, C3 and K3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-19" class="evday"> | <div id="2016-07-19" class="evday"> | ||
| Line 416: | Line 373: | ||
* Ligation and transformation in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.235_:_Cloning_protocol_for_IDT_sequences Protocol#5] and [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Ligation and transformation in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.235_:_Cloning_protocol_for_IDT_sequences Protocol#5] and [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
* Interlab studies: transformation in DH5α ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Interlab studies: transformation in DH5α ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-20" class="evday"> | <div id="2016-07-20" class="evday"> | ||
| Line 425: | Line 380: | ||
* Colony PCR on DesA-RFP, DesC-RFP ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR on DesA-RFP, DesC-RFP ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Plasmid purification gel migration DesB rbs, DesA rbs, DesB ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmid purification gel migration DesB rbs, DesA rbs, DesB ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-21" class="evday"> | <div id="2016-07-21" class="evday"> | ||
| Line 433: | Line 386: | ||
* Interlab studies: measurements plate reader | * Interlab studies: measurements plate reader | ||
| − | |||
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</div> | </div> | ||
<div id="2016-07-22" class="evday"> | <div id="2016-07-22" class="evday"> | ||
| Line 441: | Line 392: | ||
* Colony PCR on FliC, FliD, DesA DesB and CsgA ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR on FliC, FliD, DesA DesB and CsgA ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-25" class="evday"> | <div id="2016-07-25" class="evday"> | ||
| Line 451: | Line 400: | ||
* Plasmid purification DesC Csg1 DesC+RBS and digestion E/P ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmid purification DesC Csg1 DesC+RBS and digestion E/P ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
* Digestion E/P DesA+RBS, DesB ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion E/P DesA+RBS, DesB ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-26" class="evday"> | <div id="2016-07-26" class="evday"> | ||
| Line 459: | Line 406: | ||
* Competent Tg1 preparation ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol#1]). | * Competent Tg1 preparation ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.231_:_Preparation_of_competent_bacteria_cells Protocol#1]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-27" class="evday"> | <div id="2016-07-27" class="evday"> | ||
| Line 467: | Line 412: | ||
* Digestion K88005 E/S, fliC desulfo X/P, pSB1K3 E/P ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion K88005 E/S, fliC desulfo X/P, pSB1K3 E/P ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-28" class="evday"> | <div id="2016-07-28" class="evday"> | ||
| Line 476: | Line 419: | ||
* Colony PCR from respread dishes 27/7 on DesC-RBS, DesA-7.8, DesA-7.1, FliC desulfo ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR from respread dishes 27/7 on DesC-RBS, DesA-7.8, DesA-7.1, FliC desulfo ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* PCR gradient on csgA (BBa140034) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * PCR gradient on csgA (BBa140034) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-07-29" class="evday"> | <div id="2016-07-29" class="evday"> | ||
| Line 486: | Line 427: | ||
* Ligation 3A pSB1K3 RBS FliC desulfo ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation 3A pSB1K3 RBS FliC desulfo ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Redone gel migration PCR 28/7 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Redone gel migration PCR 28/7 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-08-01" class="evday"> | <div id="2016-08-01" class="evday"> | ||
| Line 495: | Line 434: | ||
* Transformation ligation 29/7 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation ligation 29/7 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
* PCR amplification IDT sequences DesA/B/C fliC coli and oligo, gel extract ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3] and [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * PCR amplification IDT sequences DesA/B/C fliC coli and oligo, gel extract ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3] and [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-08-02" class="evday"> | <div id="2016-08-02" class="evday"> | ||
| Line 509: | Line 446: | ||
[[File:T--Aix-Marseille--PCR-02-08.jpg|left|thumb|Digestion of backbones pSB1C3, pSB1A3 and pSB1K3 verifications. Like all our backbone digestions, the awaited size is 1070bp.]] | [[File:T--Aix-Marseille--PCR-02-08.jpg|left|thumb|Digestion of backbones pSB1C3, pSB1A3 and pSB1K3 verifications. Like all our backbone digestions, the awaited size is 1070bp.]] | ||
| − | |||
</div><div id="2016-08-03" class="evday"> | </div><div id="2016-08-03" class="evday"> | ||
| Line 519: | Line 455: | ||
- by 3A technical : Bba_K1951005 and Bba_K880005 digested the 07.18.2016 | - by 3A technical : Bba_K1951005 and Bba_K880005 digested the 07.18.2016 | ||
* Transformation of BBa_B0034+BBa_I0500 ligated in Tg1 and stread on Amp LB agar petri dishes ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of BBa_B0034+BBa_I0500 ligated in Tg1 and stread on Amp LB agar petri dishes ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
| − | + | </div><div id="2016-08-04" class="evday"> | |
| − | + | ||
| − | + | ||
===== Thursday, August 4th ===== | ===== Thursday, August 4th ===== | ||
| Line 529: | Line 463: | ||
* Colony PCR from the transformed bacteria of 08.03.2016 and starter of the good clones have been sent ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR from the transformed bacteria of 08.03.2016 and starter of the good clones have been sent ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Plasmid purification of DesA, clone3(141.47ng/µL), FliC <i>E. coli </i> clone 2 (169,76ng/µL) and FliC Desulfo (133,47ng/µL) valided to the sequencage have been made ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmid purification of DesA, clone3(141.47ng/µL), FliC <i>E. coli </i> clone 2 (169,76ng/µL) and FliC Desulfo (133,47ng/µL) valided to the sequencage have been made ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
| − | + | </div> | |
| − | + | ||
| − | + | ||
<div id="2016-08-05" class="evday"> | <div id="2016-08-05" class="evday"> | ||
| Line 539: | Line 471: | ||
* SLIC of CsgA in pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). | * SLIC of CsgA in pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). | ||
* Sent to the sequencing Bba_K1951009 and BBa_B0034+BBa_I0500. | * Sent to the sequencing Bba_K1951009 and BBa_B0034+BBa_I0500. | ||
| − | + | </div> | |
| − | + | ||
<div id="2016-08-08" class="evday"> | <div id="2016-08-08" class="evday"> | ||
| Line 553: | Line 484: | ||
[[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.]] | ||
| − | + | </div> | |
| − | + | ||
<div id="2016-08-09" class="evday"> | <div id="2016-08-09" class="evday"> | ||
| Line 564: | Line 494: | ||
- 98°C 30s, (98°C 10s, 60°C 30s, 72°C 1min)*30, 72°C 2min. | - 98°C 30s, (98°C 10s, 60°C 30s, 72°C 1min)*30, 72°C 2min. | ||
- 98°C 30s, (98°C 10s, 72°C 1min)*30, 72°C 2min. | - 98°C 30s, (98°C 10s, 72°C 1min)*30, 72°C 2min. | ||
| − | + | </div> | |
| − | + | ||
<div id="2016-08-10" class="evday"> | <div id="2016-08-10" class="evday"> | ||
| Line 574: | Line 503: | ||
* PCR clean up realised on PCR from the 08.09.2016, DesB (35ng/µL) and DesD (48ng/µL) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | * PCR clean up realised on PCR from the 08.09.2016, DesB (35ng/µL) and DesD (48ng/µL) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.237_:_PCR_clean-up_and_gel_extraction Protocol#7]). | ||
* SLIC realised from the previous PCR in pSB1C3 and transformation in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). | * SLIC realised from the previous PCR in pSB1C3 and transformation in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). | ||
| − | + | </div> | |
| − | + | ||
<div id="2016-08-11" class="evday"> | <div id="2016-08-11" class="evday"> | ||
| Line 584: | Line 512: | ||
* Digestion of Bba_K1951007 by PstI and XbaI at 37°C during 1h ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion of Bba_K1951007 by PstI and XbaI at 37°C during 1h ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Ligation over night of Bba_K1951007 and Bba_K880005 to create Bba_K1951010 (CsgA producer) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation over night of Bba_K1951007 and Bba_K880005 to create Bba_K1951010 (CsgA producer) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | + | </div> | |
| − | + | ||
<div id="2016-08-12" class="evday"> | <div id="2016-08-12" class="evday"> | ||
| Line 593: | Line 520: | ||
* Plasmid purification on Bba_K1951009 (116.84ng/µL) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmid purification on Bba_K1951009 (116.84ng/µL) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
* Ligation over night of Bba_K1951007 and Bba_K880005 to create Bba_K1951010 (CsgA producer) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Ligation_transformation Protocol#4]). | * Ligation over night of Bba_K1951007 and Bba_K880005 to create Bba_K1951010 (CsgA producer) ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Ligation_transformation Protocol#4]). | ||
| − | + | </div> | |
| − | + | ||
<div id="2016-08-16" class="evday"> | <div id="2016-08-16" class="evday"> | ||
| Line 603: | Line 529: | ||
* Colony PCR of Bba_K1951001 and Bba_K1951003 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR of Bba_K1951001 and Bba_K1951003 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Screening on agarose gel; 7 clones have been mixed in a PCR tube, and mix in 1µL used as DNA template. | * Screening on agarose gel; 7 clones have been mixed in a PCR tube, and mix in 1µL used as DNA template. | ||
| − | |||
</div> | </div> | ||
<div id="2016-08-17" class="evday"> | <div id="2016-08-17" class="evday"> | ||
| Line 613: | Line 538: | ||
* SLIC of DesB and DesD in pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). | * SLIC of DesB and DesD in pSB1C3 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). | ||
* Transformation of the previous SLIC in compétante Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of the previous SLIC in compétante Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
| − | |||
</div> | </div> | ||
<div id="2016-08-18" class="evday"> | <div id="2016-08-18" class="evday"> | ||
| Line 624: | Line 548: | ||
* Digestion of Bba_K1951005, Bba_K1951006, Bba_K1951005 by XbaI and PstI ; Bba_K1951007 by SpeI and PstI ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion of Bba_K1951005, Bba_K1951006, Bba_K1951005 by XbaI and PstI ; Bba_K1951007 by SpeI and PstI ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Migration of the previous biobrick (size waited : Bba_K1951005=1547pb, Bba_K1951006=944pb, Bba_K1951007=506pb). | * Migration of the previous biobrick (size waited : Bba_K1951005=1547pb, Bba_K1951006=944pb, Bba_K1951007=506pb). | ||
| − | |||
</div> | </div> | ||
<div id="2016-08-19" class="evday"> | <div id="2016-08-19" class="evday"> | ||
| Line 634: | Line 557: | ||
* Colony PCR on the SLIC transformation of the 08.17.2016, nothing has worked ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR on the SLIC transformation of the 08.17.2016, nothing has worked ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* Sequencing of the Bba_K1951004 is ok (desA producer). | * Sequencing of the Bba_K1951004 is ok (desA producer). | ||
| − | |||
</div> | </div> | ||
<div id="2016-08-22" class="evday"> | <div id="2016-08-22" class="evday"> | ||
| Line 644: | Line 566: | ||
* SLIC and Transformation of Bba_K1951005 and Bba_K1951001 in competant Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_7_:_SLIC_.28sequence-_and_ligation-independent_cloning.29 Protocol#10]). | * SLIC and Transformation of Bba_K1951005 and Bba_K1951001 in competant 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> | ||
<div id="2016-08-23" class="evday"> | <div id="2016-08-23" class="evday"> | ||
| Line 655: | Line 576: | ||
* Colony PCR from Bba_K1951008, Bba_K1951009 | * Colony PCR from Bba_K1951008, Bba_K1951009 | ||
* Starters from Bba_K1951008 and Bba_K1951009 that seem ok. | * Starters from Bba_K1951008 and Bba_K1951009 that seem ok. | ||
| − | |||
</div> | </div> | ||
<div id="2016-08-24" class="evday"> | <div id="2016-08-24" class="evday"> | ||
| Line 663: | Line 583: | ||
* Plasmid purification from Bba_K1951008, Bba_K1951009 and Bba_K1951001 and send to the sequencing with oligo FW ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | * Plasmid purification from Bba_K1951008, Bba_K1951009 and Bba_K1951001 and send to the sequencing with oligo FW ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Plasmid_DNA_purification Protocol#4]). | ||
* Colony PCR from Bba_K1951005, Bba_K1951008, Bba_K1951009 and Bba_K1951010 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR from Bba_K1951005, Bba_K1951008, Bba_K1951009 and Bba_K1951010 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | |||
</div> | </div> | ||
<div id="2016-08-25" class="evday"> | <div id="2016-08-25" class="evday"> | ||
| Line 700: | Line 619: | ||
* 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]). | ||
* Ligation of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) + BBa_I0500 (Prom) 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) + BBa_I0500 (Prom) overnight at 16°C ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
| − | |||
</div> | </div> | ||
<div id="2016-08-29" class="evday"> | <div id="2016-08-29" class="evday"> | ||
| Line 710: | Line 628: | ||
* 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. | ||
| − | |||
</div> | </div> | ||
<div id="2016-08-30" class="evday"> | <div id="2016-08-30" class="evday"> | ||
| Line 725: | Line 642: | ||
[[File:T--Aix-Marseille--PCR-30-06.jpg|left|thumb|Migration of FliC from E.coli, FliC from desulfo, DesB and DesC. The awaited size for the 8 DesD+RBS is 2150bp, for the FliC 25-54 it's 1900bp.]] | [[File:T--Aix-Marseille--PCR-30-06.jpg|left|thumb|Migration of FliC from E.coli, FliC from desulfo, DesB and DesC. The awaited size for the 8 DesD+RBS is 2150bp, for the FliC 25-54 it's 1900bp.]] | ||
| − | |||
</div> | </div> | ||
<div id="2016-09-01" class="evday"> | <div id="2016-09-01" class="evday"> | ||
| Line 741: | Line 657: | ||
* Transformation of Bba_K1951008 in fliC mutant Keio competante made the 30th, spread on Kana/Cm petri dishes ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of Bba_K1951008 in fliC mutant Keio competante 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> | ||
<div id="2016-09-02" class="evday"> | <div id="2016-09-02" class="evday"> | ||
| Line 753: | Line 667: | ||
- 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 | ||
- RBS Bba_K1951002(E/S) and RBS Bba_K1951003(X/P) in pSB1K3(E/P) at 16°C overnight | - RBS Bba_K1951002(E/S) and RBS Bba_K1951003(X/P) in pSB1K3(E/P) at 16°C overnight | ||
| − | |||
</div> | </div> | ||
<div id="2016-09-05" class="evday"> | <div id="2016-09-05" class="evday"> | ||
| Line 788: | Line 701: | ||
* Colony PCR to verify the insertion of Bbs1 site in the 214-215 and 238-239 sites of BBa_K1951008 and BBa_K1951009 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR to verify the insertion of Bbs1 site in the 214-215 and 238-239 sites of BBa_K1951008 and BBa_K1951009 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
* From a starter of intermediate biobrick BBa_K1951004+ RBS BBa_K1951001, made culture at DO=0.2 until DO=0.6 and took a sample of 1uDO. Added 50µL beta mercatoethanol to the sample and congeled at -20°C to test protein production on a SDS page/Comassie. | * From a starter of intermediate biobrick BBa_K1951004+ RBS BBa_K1951001, made culture at DO=0.2 until DO=0.6 and took a sample of 1uDO. Added 50µL beta mercatoethanol to the sample and congeled at -20°C to test protein production on a SDS page/Comassie. | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-09-08" class="evday"> | <div id="2016-09-08" class="evday"> | ||
| Line 800: | Line 711: | ||
* 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 competant Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of the previous ligation in competant 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"> | ||
| Line 811: | Line 721: | ||
* Digestion test by BBs1 and EcoRI using NEB buffer and electrophoresis analyse ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Digestion test by BBs1 and EcoRI using NEB buffer and electrophoresis analyse ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Colony PCR of BBa_K1951011 in pSB1C3 by amplification with different SLIC oligos FW and RV to see the differente constitutive biobrick. Biobrick is OK ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | * Colony PCR of BBa_K1951011 in pSB1C3 by amplification with different SLIC oligos FW and RV to see the differente constitutive biobrick. Biobrick is OK ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_PCR_and_electrophoresis Protocol#3]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-09-10" class="evday"> | <div id="2016-09-10" class="evday"> | ||
| Line 820: | Line 728: | ||
* Swimming test of the knockout fliC mutant W3110 for 10 differents colonies and the W3110 WT as a positive temoin ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.2311_:_Swimming_test Protocol#11]). | * Swimming test of the knockout fliC mutant W3110 for 10 differents colonies and the W3110 WT as a positive temoin ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.2311_:_Swimming_test Protocol#11]). | ||
* Starters of the fliC mutant W3110. | * Starters of the fliC mutant W3110. | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-09-11" class="evday"> | <div id="2016-09-11" class="evday"> | ||
| Line 830: | Line 736: | ||
* Transformation of the previous competant with BBa_K1951008 and BBa_K1951009 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of the previous competant 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> | ||
<div id="2016-09-12" class="evday"> | <div id="2016-09-12" class="evday"> | ||
| Line 838: | Line 742: | ||
* Transformation of the fliC mutant W3110 competant with BBa_K1951008 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation of the fliC mutant W3110 competant 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"> | ||
| Line 847: | Line 750: | ||
* Monitoring growth from differents strain to make comassie. From starters of fliC mutant W3110 BBa_K1951008, liC mutant W3110 BBa_K1951009, Tg1 BBa_K1951010, Tg1 WT (negative temoin), Tg1+pSB1C3 containing RFP (negative temoin), and cadA mutant BBa_K1951011, we made cultures at DO=0.2 in duplicate. At DO=0.6, half of the cultures has been induced with 0.02 arabinose for the biobrick containing an inductible promotor. Every hour, a sample of 1uDO was taken. After centrifugation at 5000g during 5min and removing of the supernatant, we added 50µL beta mercatoethanol and congeled at -20°C to test protein production on a SDS page/Comassie later. | * Monitoring growth from differents strain to make comassie. From starters of fliC mutant W3110 BBa_K1951008, liC mutant W3110 BBa_K1951009, Tg1 BBa_K1951010, Tg1 WT (negative temoin), Tg1+pSB1C3 containing RFP (negative temoin), and cadA mutant BBa_K1951011, we made cultures at DO=0.2 in duplicate. At DO=0.6, half of the cultures has been induced with 0.02 arabinose for the biobrick containing an inductible promotor. Every hour, a sample of 1uDO was taken. After centrifugation at 5000g during 5min and removing of the supernatant, we added 50µL beta mercatoethanol and congeled at -20°C to test protein production on a SDS page/Comassie later. | ||
* HPLC test of the fliC mutant complemented with BBa_K1951011 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.239_:_Cadaverin_HPLC_analysis Protocol#9]). | * HPLC test of the fliC mutant complemented with BBa_K1951011 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.239_:_Cadaverin_HPLC_analysis Protocol#9]). | ||
| − | |||
| − | |||
</div> | </div> | ||
<div id="2016-09-15" class="evday"> | <div id="2016-09-15" class="evday"> | ||
| Line 858: | Line 759: | ||
* Ligation of 5ng from the annealing mix for 100ng pSB1C3 digested by EcoRI and PstI ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | * Ligation of 5ng from the annealing mix for 100ng pSB1C3 digested by EcoRI and PstI ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.233_:_Cloning_protocol_for_IDT_sequences Protocol#5]). | ||
* Transformation in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | * Transformation in Tg1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.232_:_Transformations Protocol#2]). | ||
| − | |||
| − | |||
</div> | </div> | ||
{{:Team:Aix-Marseille/Template-Footer}} | {{:Team:Aix-Marseille/Template-Footer}} | ||
