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)
Tuesday, August 16th
Sequencing of Bba_K1951002 has been confirmed as ok* Miniprep on Bba_K1951009 (116.84ng/µL)
Colony PCR of Bba_K1951001 and Bba_K1951003
Screening on agarose gel; 7 clones have been mixed in a PCR tube, and mix in 1µL used as DNA template
Wednesday, August 17th
Made petri dishes (10 50µG/mL kanamycin and 10 50µG/mL ampicillin)
Colony PCR of Bba_K1951007
SLIC of DesB and DesD in pSB1C3
Transformation of the previous SLIC in compétante Tg1
Thursday, August 18th
Miniprep of Bba_K1951007(203.18ng/µL), Bba_K1951003(187.53ng/µL), Bba_K1951001 (188.74ng/µL)
Glyceroled tubes on the previous biobricks
Previous biobrick sent to the sequencing
Digestion of Bba_K1951005, Bba_K1951006, Bba_K1951005 by XbaI and PstI ; Bba_K1951007 by SpeI and PstI
Migration of the previous biobrick (size waited : Bba_K1951005=1547pb, Bba_K1951006=944pb, Bba_K1951007=506pb
Friday, August 19th
Ligation of the digestion from the 08.18.2016 using 3A method in both pSB1A3 and pSB1C3 at room temperature during 1h
Transformation of the previous ligation in Tg1
Colony PCR on the SLIC transformation of the 08.17.2016, nothing has worked.
Sequencing of the Bba_K1951004 is ok (desA producer)
Monday, August 22th
PCR using Q5 master with new FliC coli sequence ( substitute Bbs1 site)Bba_K1951005 and Bba_K1951001, every SLIC have been validated by electrophoresis
PCR clean up on the 2 previous SLIC
SLIC and Transformation of Bba_K1951005 and Bba_K1951001 in competant Tg1
Starter of Bba_K1951004, Bba_K1951009 and Bba_K1951010 to make a protein production test
Tuesday, August 23th
Colony PCR from transformation of the 08.25.2016
From starter of the 08.22.2016, culture was started at A(600nm) = 0.1 and induced with 0.02% arabinose at A(600nm)=0.5. After 2h incubation, DO was taken again and 1uDO was sampled. The sample was centrifiged at 5000g during 5min. Supernant was removed and pellet resuspended in 50µL of charge buffer. 5µL was charged on a polyacrylamide gel.
Miniprep from Bba_K1951003 (171ng/µL), Bba_K1951004(217ng/µL), Bba_K1951007(139ng/µL)
Colony PCR from Bba_K1951008, Bba_K1951009
Starters from Bba_K1951008 and Bba_K1951009 that seem ok
Wednesday, August 24th
Miniprep from Bba_K1951008, Bba_K1951009 and Bba_K1951001 and send to the sequencing with oligo FW
Colony PCR from Bba_K1951005, Bba_K1951008, Bba_K1951009 and Bba_K1951010
Thursday, August 25th
Digestion of Bba_K1951002 and Bba_K1951003 by XbaI and PstI, Bba_B0034 by SpeI and PstI
Friday, August 26th
Sequencing Bba_K1951009, Bba_K1951001, Bba_K1951008 and Bba_K1951010 ok with the FW oligo only
Preparation of the HPLC solution :
- Buffer Borate 0.05M pH9 ( ajusted with NaOH)
- L-Lysine 0.01M
- DEEMM 0.2M
- Hexamethylenediamine 0.01M
Test of the HPLC C18 column with 3 mixs :
1 (Lysine): Borate 180µL, Methanol 60µL, H2O ppi 47µL, Lysine 30µL, DEEMM 3µL
2 (Hexamethylenediamine): Borate 180µL, Methanol 60µL, H2O ppi 47µL, Hexamethylenediamine 30µL, DEEMM 3µL
3 (Negative temoin): Borate 180µL, Methanol 60µL, H2O ppi 77µL, DEEMM 3µL
Heat to 70°C during 2 hours
Pcr clean up on the digestion from the 08.25.2016
Ligation using 2A method of Bba_K1951002 or Bba_K1951003 and Bba_B0034
Transformation of the previous ligation in competant Tg1
Digestion of 500ng in 50µL total mix of Bba_K1951002 and Bba_K1951005 by XbaI and PstI
