Monday, June 13th
- Preparation of heat competent DH5α cells
Tuesday, June 14th
- Transformation of DH5α cells with test plasmid
Wednesday, June 15th
- Preparation of heat competent Tg1 cells
- Transformation of Tg1 cells with test plasmid
Thursday, June 16th
- Transformation of DH5α cells with pSB1C3 + RFP
- Transformation with K880005, K823017, I0500, K1404006, B0034
Friday, June 17th
- Miniprep K880005, K823017, I0500, K1404006, B0034
- Miniprep pSB1C3 + RFP
Monday, June 20th
- pSB1C3 digestion from 17/06 miniprep
- pSB1C3 digestion gel migration : NO PHOTO
- Biobricks minipreps gel migration : OK PHOTO
- Transformation Tg1 with RFP+pSB1C3
Tuesday, June 21th
- pSB1C3 digestion and gel migration, NucleoSpin gel extract and PCR cleanup
- FliC E.Coli and FliC Desulfo digestion E/P and ligation with pSB1C3
- E. Coli and Desulfo FliC ligation in linearized pSB1C3
- Transformation of pSB1C3 RFP in Tg1
Wednesday, June 22th
- Transformation in DH5α of :
- FliC coli/desulfo in pSB1C3
- Linearized pSB1K3
- Linearized pSB1A3
- Linearized pSB1T3
- Linearized pSB1C3
- Miniprep B0034
- Transformation in DH5α of interlab studies plasmids
Thursday, June 23th
- Digestion E/P pSB1C3
- Purification pSB1C3 purified linearized 23/06
- Digestion E/P of DesA, DesB, DesC, DesD
- 3A Assembly of B0034 and I0500 in pSB1K3
- Digestion with pSB1C3 23/06 insert digested fliC
- Transformation in Tg1 and DH5α
Friday, June 24th
- Digestion E/P pSB1C3, pSB1A3, pSB1K3, pSB1T3, and agarose gel extract
- Colony PCR of
- Ligation: FliC E.Coli, FliC desulfo, DesA B C D
- Ligation pSB1C3 and fliC E.Coli / desulfo digested E/P 21/06
- Transformation in Tg1 and DH5α
- Redone transformation test device 1 and 3 interlab studies
- Miniprep pSB1C3 pSB1CA3 pSB1CK3 pSB1CT3
Monday, June 27th
- Transformations DesA, DesB, DesC, DesD, FliC E.Coli and desulfo, interlab studies
- Transformations failed
- Transformation pSB1T3 miniprep, pSB1K3 miniprep, control
Tuesday, June 28th
- Miniprep promoter+RBS
- PCR of IDT sequences (fliC and DesA to D) and gel check
- Gel extract pSB1CK3, pSB1CC3, pSB1CT3
Wednesday, June 29th
- Miniprep from interlab studies, T3 and K3
- Ligation and transformation in Tg1 Sandra Des A to D, fliC coli desulfo
- Redone PCR of IDT sequences (fliC and DesA to D) (different Tm) and gel extract
- Transformation of fliCs from remaining ligation in Tg1 Sandra
Thursday, June 30th
- Migration FliC coli, desulfo, DesB, DesC
Friday, July 1st
- Digestion PCR products and vector pSB1C3
- PCR clean up on digested pSB1C3, fliC coli desulfo, DesB and DesC, migration and transformation in Tg1
Monday, July 4th
- Isolated white colonies on dishes
Tuesday, July 5th
- Reisolated colonies from 04/07
Wednesday, July 6th
- Colony PCR with FLiC E.Coli Desulfo DesB DesC
Thursday, July 7th
- Miniprep: DesC DesB FliC coli
- PCR on A2 A4 B1 clones from 6/7
- Digestion E/P of A5 B4 C3 C2 minipreps and pSB1A3, pSB1C3, pSB1K3, pSB1T3
Friday, July 8th
- Transformation BOO34 and I0500 in pSB1K3
- Gel verification of 7/7 digestion
- Miniprep A5 (fliC coli) and C2 (DesB) from 7/7 starters
- PCR clean up of vectors
- PCR SLIC FliC coli/desulfo, DesA, B, C, D, csgA -> failed
Monday, July 11th
- Redone PCR SLIC 8/8 -> ok
- Redone PCR DesC, DesD and csgA
- SLIC desA, desB, fliC coli desulfo in pSB1A3
- Transformation Tg1 with fliC coli desulfo, desA, desB in pSB1A3 from SLIC
- PCR clean up desA, desB, fliC coli desulfo
Tuesday, July 12th
- Purification clean up DesC and CsgA from PCR
- Digestion E/P pSB1A3, pSB1K3, pSB1C3, PCR clean up
- Colony PCR SLIC 11/07 FliC desulfo, desA, desB
- SLIC in pSB1C3 of DesA/B/C, FliC coli and desulfo, csgA
- Transformation SLIC mix in Tg1
- Transformation constructions #7 (kanamycin) 9 and 55 (chloramphenicol)
Wednesday, July 13th
- Miniprep DesA, DesB, fliC desulfo
- Digestion and PCR clean up of pSB1C3 E/P, B0034 E/S, K88005 S/P, fliC desulfo X/P, desA X/P, desB X/P
- Colony PCR #1 2 3 50 51 53
- Miniprep pSB1 A/C/K/T 3, gel migration
Monday, July 18th
- Ligation (3A) in pSB1C3 of desA+RBS, desB+RBS, fliC desulfo+RBS
- Transformation in Tg1
- Miniprep empty vectors C3 A3 K3
- Interlab studies : resuspended FITC, incubation 42°C
- Digestion E/P and PCR clean up empty vectors A3 C3 K3
- PCR clean up …
Tuesday, July 19th
- Colony PCR …
- Ligation and transformation in Tg1
- Interlab studies: transformation in DH5α
Wednesday, July 20th
- Colony PCR …
- Miniprep gel migration DesB rbs, DesA rbs, DesB
Thursday, July 21th
- Interlab studies: measurements plate reader
Friday, July 22th
- Colony PCR … and agarose gel
Monday, July 25th
- Redone 22/7 Colony PCR … and agarose gel
- Miniprep DesC Csg1 DesC+RBS and digestion E/P
- Digestion E/P DesA+RBS, DesB
Tuesday, July 26th
- Competent Tg1 preparation
Wednesday, July 27th
- Digestion K88005 E/S, fliC desulfo X/P, pSB1K3 E/P
Thursday, July 28th
- Colony PCR from respread dishes 27/7, …
- PCR gradient on csgA (BBa140034), gel
Friday, July 29th
- Miniprep DesA DesC, E/P digestion
- Ligation 3A pSB1K3 RBS FliC desulfo
- Redone gel migration PCR 28/7
Monday, August 1st
- Transformation ligation 29/7
- PCR amplification IDT sequences DesA/B/C fliC coli and oligo, gel extract
Tuesday, August 2rd
- Colony PCR of CsgA from the 01/08 using EconoTac
- PCR clean up of DesC(200ng/µL) and gel extraction of DesA (83ng/µL), DesB(53ng/µL), FliC E. coli
- SLIC of DesA, DesB, DesC, DesD and transformation of 10µL in 100µL competant Tg1
- Sequencage of Desulfovribrio clone 3 validated
Wednesday, August 3th
- Digestion Bba_B0034 by EcoRI/XbaI (pSB1A3) and Bba_I0500 by EcoRI/(pSB1C3)
- Ligation overnight at 16°C
- by 2A technical : BBa_B0034+BBa_I0500
- 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
Thursday, August 4th
- Transformation of Bba_K1951008 (FliC E. coli and spread on kanamycin LB agar petri dishes
- From SLIC of the 08.02.2016 and 2 ligations, colonies of DesB and DesC have been repatched on chloramphenicol LB agar petri dishes
- Colony PCR from the transformed bacteria of 08.03.2016 and starter of the good clones have been sent.
- Miniprep of DesA, clone3(141.47ng/µL), FliC E. coli clone 2 (169,76ng/µL) and FliC Desulfo (133,47ng/µL) valided to the sequencage have been made.
Friday, August 5th
- Colony PCR from the transformed bacteria of 08.03.2016 and starter of the good clones have been sent.
- SLIC of CsgA in pSB1C3
- Sent to the sequencing Bba_K1951009 and BBa_B0034+BBa_I0500
Monday, August 8th
- Ligation of BBa_B0034+BBa_I0500 and Bba_K1951000 into pSB1K3 to create desA producer
- Transformation of the previous ligation in competant Tg1
- Starters from DesC and Bba_K1951008 of the 08.04.2016¨
- Resuspension of oligo using the IDT protocole
- PCR of DesB and DesC (GC enhencer, Q5 master Mix and gel extract on the good strips
- Colony PCR of CsgA, and gel electrophoresis
Tuesday, August 9th
- Colony PCR from transformation of the 08.08.2016 (size waited : 3000pb), clone1 has been sent to the sequencing
- Miniprep of Bba_K1951008 (126.52ng/µL), Bba_K1951002 (102.18ng/µL), Bba_K1951007 (8,33ng/µL)
- From the IDT sequence and gel extract of the 08.08.2016, PCR using Q5 master Mix with 2 different programs on DesB and DesD
- 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
Wednesday, August 10th
- Miniprep of Bba_K1951004 (248,49ng/µL) in pSB1K3 and sent to sequencing
- Glyceroled tubes of Bba_K1951004 (248,49ng/µL) in pSB1K3
- PCR clean up realised on PCR from the 08.09.2016, DesB (35ng/µL) and DesD(48ng/£µL)
- SLIC realised from the previous PCR in pSB1C3 and transformation in Tg1
Thursday, August 11th
- Sequencing of BBa_B0034+BBa_I0500 and Bba_K1951007 have been confirmed as ok
- Colony PRC from 08.10.2016 transformation and any clones were migrated to the good size
- Digestion of Bba_K1951007 by PstI and XbaI at 37°C during 1h
- Ligation over night of Bba_K1951007 and Bba_K880005 to create Bba_K1951010 (CsgA producer)
Friday, August 12th
- Colony PRC from 08.10.2016 transformation again (DesB and DesD)and any clones were migrated to the good size again
- Miniprep on Bba_K1951009 (116.84ng/µL)
- Ligation over night of Bba_K1951007 and Bba_K880005 to create Bba_K1951010 (CsgA producer)
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