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
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
Ligation of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) overnight at 16°C
Ligation of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) + BBa_I0500 (Prom) overnight at 16°C
Monday, August 29th
Sequencing Bba_K1951001, Bba_K1951005, Bba_K1951009 and Bba_K1951010 ok with the RV oligo only so VALIDATE.
Digestion of 500ng in 50µL total mix of Bba_K1951004 EcoRI and PstI
Ligation of Bba_K1951004 digested E/P in pSB1C3 at 16°C overnight
NB : we wanted to transform a cadA mutant from KEIO bank so we can't use this biobrick in pSB1K3.
Tuesday, August 30th
Colony PCR of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs)
Colony PCR of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) + BBa_I0500 (Prom)
Transformation of Bba_K1951004 (DesA with pSB1C3) in competant Tg1 cells
Ligation of Bba_K1951004 digested E/P in pSB1C3 at 16°C overnight
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)
Made competante cells Tg1, culture from an over night starter have grown up from Abs(600nm)=0.05 until Abs(600nm)=0.457 at 37°C
Thursday, September 1st
Miniprep of the starter Bba_K1951001(175ng/µL), Bba_K1951002(250ng/µL), Bba_K1951003(440ng/µL) and Bba_K1951005(183ng/µL)
Glyceroled tubes from starter Bba_K1951001(175ng/µL), Bba_K1951002(250ng/µL), Bba_K1951003(440ng/µL) and Bba_K1951005(183ng/µL)and from starter of cadA mutant strain and fliC mutant strain from Keio bank.
Digestion of intermediate biobricks BBa_K1951004 by SpeI/PstI ; Bba_K1951001 (DesB) and Bba_K1951003(DesD)with BBa_B0034 (RBS) by XbaI, PstI ; Bba_K1951002 (DesC) with Bba_B0034 (RBS) by EcoRI/SpeI
Ligation of Bba_K1951004 (S/P) and Bba_K1951001 + RBS (X/P) in pSB1K3(E/P) at 16°C overnight
Ligation of Bba_K1951002 + RBS (E/S) and Bba_K1951003 + RBS (X/P) in pSB1K3(E/P) at 16°C overnight
Made competante cell of fliC mutant and cadA mutant from the Keio bank and test on different antibiotic petri dishes (only kanamycin dish was a positive control)
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
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 competante from 08.30.2016
Transformation of Bba_K1951008 in fliC mutant Keio competante made the 30th, spread on Kana/Cm petri dishes
Made a calibration range for HPLC
Friday, September 2rd
Remade ligation of pSB1C3 (E/P) and Bba_K1951004
Transformation of the previous ligation mix in competant Tg1
Colony PCR on transformation of intermediate biobricks :
- 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
Monday, September 5th
Analyse of the electrophoresis results from 09.02.2016
Swimming test of the WT Keio strain and the fliC mutant keio strain. Nor the WT neither the fliC mutant strain are abled to swim. We decide to make a fliC mutant from a good swimmer strain (Escherichia coli W3110)
Colony PCR 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
Tuesday, September 6th
Transformation of cadA mutant competant strain by Bba_K1951004 in pSB1C3, spread on Kana/Cm petri dishes
Colony PCR 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
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.
Transformation of the previous PCR in competant Tg1
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
Wednesday, September 7th
Colony PCR on cadA mutant complemented with BBa_K1951004
HPLC test of cadA mutant complemented with BBa_K1951004
Colony PCR to verify the insertion of Bbs1 site in the 214-215 and 238-239 sites of BBa_K1951008 and BBa_K1951009
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
Thursday, September 8th
Starters of BBa_K1951008 + BbsI 215-216 position and starters of BBa_K1951008 + BbsI 238-239 position
Spreading of transduction in a E. coli W3110 strain using the phage P1 from the 09.05.2016 (500£µL cells + 2µL P1 or 10µL P1 or 10µL P1 or 50µL P1 or 100µL P1)
Digestion of intermediate biobrick BBa_K1951004+ RBS BBa_K1951001 by EcoRI and SpeI and RBS BBa_K1951001+ RBS BBa_K1951002 by SpeI and PstI
Ligation of the 2 previous digestions in pSB1C3 to make the final BBa_K1951011 (E/P)
Transformation of the previous ligation in competant Tg1
Friday, September 9th
Derivatization of the sample cadA mutant complemented with BBa_K1951004 and cadA mutant complemented with BBa_K1951004+ RBS BBa_K1951001
Reisolement of the knockout fliC mutant W3110 from the transformation of the 09.08.2016
Miniprep of the BBa_K1951008 + BbsI 215-216 position and starters of BBa_K1951008 + BbsI 238-239 position
Digestion test by BBs1 and EcoRI using NEB buffer and electrophoresis analyse
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.
Satursday, September 10th
Swimming test of the knockout fliC mutant W3110 for 10 differents colonies and the W3110 WT as a positive temoin.
Starters of the fliC mutant W3110
Monitoring growth from differents strain to make comassie. From starters of biobrick BBa_K1951008, BBa_K1951009, BBa_K1951010, Tg1 WT (negative temoin), Tg1+pSB1C3 containing RFP (negative temoin), and 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 centrigugation 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.
Sunday, September 11st
Made competant of the fliC mutant W3110 strain from 5 differents colonies from 4 colonies which swam well
Transformation of the previous competant with BBa_K1951008 and BBa_K1951009
Transformation of cadA mutant Keio strain with BBa_K1951011
Monday, September 12rd
Transformation of the fliC mutant W3110 competant with BBa_K1951008
Tuesday, September 13th
Swimming test of colonies obtained after transformation of the 09.12.2016. After 3h incubation at 37°C, swimming has been recovered
HPLC test of the fliC mutant complemented with BBa_K1951011
Thursday, September 15
Dillution of peptides oligos by 10000 (ex: 0.31mg/238µL/10000=ng/µL
Annealing 98°C during 2min and cool down during approximatly 3h
Ligation of 5ng from the annealing mix for 100ng pSB1C3 digested by EcoRI and PstI
