* 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]).
- 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 (Protocol#2).
Thursday, August 4th
Transformation of Bba_K1951008 (FliC E. coli and spread on kanamycin LB agar petri dishes (Protocol#2).
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 (Protocol#3).
Plasmid purification 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 (Protocol#4).
Friday, August 5th
Colony PCR from the transformed bacteria of 08.03.2016 and starter of the good clones have been sent (Protocol#3).
Transformation of the previous SLIC in compétante Tg1 (Protocol#2).
Thursday, August 18th
Plasmid purification of Bba_K1951007(203.18ng/µL), Bba_K1951003(187.53ng/µL), Bba_K1951001 (188.74ng/µL) (Protocol#4).
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 (Protocol#5).
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 (Protocol#5).
Transformation of the previous ligation in Tg1 (Protocol#2).
Colony PCR on the SLIC transformation of the 08.17.2016, nothing has worked (Protocol#3).
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 (Protocol#3).
SLIC and Transformation of Bba_K1951005 and Bba_K1951001 in competant Tg1 (Protocol#10).
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 (Protocol#3).
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.
Plasmid purification from Bba_K1951003 (171ng/µL), Bba_K1951004(217ng/µL), Bba_K1951007(139ng/µL) (Protocol#4).
Colony PCR from Bba_K1951008, Bba_K1951009
Starters from Bba_K1951008 and Bba_K1951009 that seem ok.
Wednesday, August 24th
Plasmid purification from Bba_K1951008, Bba_K1951009 and Bba_K1951001 and send to the sequencing with oligo FW (Protocol#4).
Colony PCR from Bba_K1951005, Bba_K1951008, Bba_K1951009 and Bba_K1951010 (Protocol#3).
Thursday, August 25th
Digestion of Bba_K1951002 and Bba_K1951003 by XbaI and PstI, Bba_B0034 by SpeI and PstI (Protocol#5).
Friday, August 26th
Sequencing Bba_K1951009, Bba_K1951001, Bba_K1951008 and Bba_K1951010 ok with the FW oligo only.
Pcr clean up on the digestion from the 08.25.2016 (Protocol#7).
Ligation using 2A method of Bba_K1951002 or Bba_K1951003 and Bba_B0034 (Protocol#5).
Transformation of the previous ligation in competant Tg1 (Protocol#2).
Digestion of 500ng in 50µL total mix of Bba_K1951002 and Bba_K1951005 by XbaI and PstI (Protocol#5).
Ligation of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) overnight at 16°C (Protocol#5).
Ligation of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) + BBa_I0500 (Prom) overnight at 16°C (Protocol#5).
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 (Protocol#5).
Ligation of Bba_K1951004 digested E/P in pSB1C3 at 16°C overnight (Protocol#5).
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) (Protocol#3).
Colony PCR of Bba_K1951001(DesB), Bba_K1951002(DesC), Bba_K1951003(DesD) and Bba_B0034 (rbs) + BBa_I0500 (Prom) (Protocol#3).
Transformation of Bba_K1951004 (DesA with pSB1C3) in competant Tg1 cells (Protocol#2).
Ligation of Bba_K1951004 digested E/P in pSB1C3 at 16°C overnight (Protocol#5).
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 (Protocol#1).
Thursday, September 1st
Plasmid purification of the starter Bba_K1951001(175ng/µL), Bba_K1951002(250ng/µL), Bba_K1951003(440ng/µL) and Bba_K1951005(183ng/µL) (Protocol#4).
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 (Protocol#5).
Ligation of Bba_K1951004 (S/P) and Bba_K1951001 + RBS (X/P) in pSB1K3(E/P) at 16°C overnight (Protocol#5).
Ligation of Bba_K1951002 + RBS (E/S) and Bba_K1951003 + RBS (X/P) in pSB1K3(E/P) at 16°C overnight (Protocol#5).
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) (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 (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 competante from 08.30.2016 (Protocol#2).
Transformation of Bba_K1951008 in fliC mutant Keio competante made the 30th, spread on Kana/Cm petri dishes (Protocol#2).
Made a calibration range for HPLC.
Friday, September 2rd
Remade ligation of pSB1C3 (E/P) and Bba_K1951004 (Protocol#5).
Transformation of the previous ligation mix in competant Tg1 (Protocol#2).
Colony PCR on transformation of intermediate biobricks (Protocol#3).:
- 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
Analysis 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) (Protocol#11).
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 (Protocol#3).
Transformation of the previous PCR in competant Tg1 (Protocol#2).
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 (Protocol#3).
PCR colony verification.
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 (Protocol#5).
Ligation of the 2 previous digestions in pSB1C3 to make the final BBa_K1951011 (E/P) (Protocol#5).
Transformation of the previous ligation in competant Tg1 (Protocol#2).
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
Plasmid purification of the BBa_K1951008 + BbsI 215-216 position and starters of BBa_K1951008 + BbsI 238-239 position (Protocol#4).
Digestion test by BBs1 and EcoRI using NEB buffer and electrophoresis analyse (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.
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.
Sunday, September 11st
Made competant of the fliC mutant W3110 strain from 5 differents colonies from 4 colonies which swam well (Protocol#1).
Transformation of the previous competant with BBa_K1951008 and BBa_K1951009 (Protocol#2).
Transformation of cadA mutant Keio strain with BBa_K1951011 (Protocol#2).
Monday, September 12rd
Transformation of the fliC mutant W3110 competant with BBa_K1951008 (Protocol#2).
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.
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
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 (Protocol#5).
