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Team:Hannover/Notebook

Notebook

date activity summary
04-21-16 Bianca preparation of working area pipets and other stuff autoclaved
04-28-16 Janis, Bianca testing of pipets pipets work well
assembly of TALEs we cloned three different constructs in TALEs and transformed them into E.coli: 1) R-SCTALEN left HA Tag 2) R-TALEN left HA Tag 3) R-TALEN right myc Tag
isolation of plasmids We isolated the two plasmids 1) iGEM20? and 2) pET 32a+? out of E.coli with a QIAprep(R) Spin Miniprep Kit. The concentrations were 1) 354 ng/ µl and 2) 43.5 ng/ µl dsDNA measured with a NanoPhotometer
05-03-16 Janis assembly of TALEs, 2nd try Cells could not be transformed, propably GGC of vectors did not work. Did the assembly again, this time over night.
PCR/Gels/Prep/Cloning Primers arrived. Did PCR with N454-F/R from N454-HA and TRX_mod-F/R from PET32. PCR products cloned into PUC57-vector
05-23-16 Bianca plasmid isolation Plasmid isolation with QIAprep Spin Miniprep kit. The protocol attached to the kit was used without step 7.
Plasmids: TRXmodule1 (1), TRXmodule2 (2), iGEMN454-1 (3), iGEMN454-2 (4), 1-1, 1-2, 2-2, 3-1, 3-2, 5 FOK DS wos (without stop), 6 FOK RR wos, 7 Sc FOK wos
control digest Control digest with plasmids (5) - (9). Mastermix for (5)-(9): 3 µl EcoRI-HF, 3 µl BamHI-HF, 12 µl buffer CutSmart and 66 µl H2O, 14 µl of the Mastermix were added to 6 µl of each plasmid DNA
Control digest with plasmids (1) - (4). Mastermix for (1)-(4): 5 µl BsaI 10 µl CutSmart, 55 µl H22O, 14 µl of the Mastermix were added to 6 µl of plasmid DNA
The digests ran for 1 h at 37 °C
agarose gel After the digest 5 µl loadingbuffer was added to the control digest, 20 µl were loaded on a 1 % agarose gel for 30 min and at 140 V. 7 µl of a 1 kb DNA ladder were used.
05-25-16 Janis sequencing Plasmids and primer were pipetted.
05-26-16 Bianca TNF reaction 500 ng DNA, 1 µl Met, ad 50 µl H2O. Used DNA: iGEM 1-2 (A and B), MPAx SCL, 90 min at 30 °C
in-vitro-cleavage assay TC(-): 12,5 µl DNA, 2 µl enzyme, 2 µl NEB3 buffer, 3,5 µl H2O (+) Ax7: 10 µl DNA, 2 µl enzyme, 2 µl NEB3 buffer, 6 µl H2O; 30 min at 37 °C
MiniElute PCR Purification Kit, 100 µl buffer PB, probes frozen with glycerin
05-30-16 Bianca in-vitro-cleavage assay 2 µl DNA ((+)AX7 und TC(-)), 3 µl NEB3 buffer, 5 µl BSA, 4 µl TNT(-)/Scl + 16 µl H2O or 13 µl TNT(+) + 7 µl HH2OO, ad 30 µl HH2OO
centrifugation for: 1 h at 37 °C, 20 min at 65 °C, 10 min at 7000xg, 3 min at 14000xg, 20 µl supernatant + 5 µl LB
agarose gel 1 % agarose gel, 50 ml liquide agarose + 5 µl RedSafe, 20 min at 140 V
Western blot/preparing probes at 37 °C, 30- (4 µl) and 30+ (13 µl) with 60 µl Laemmli, after 0, 30, 90 and 180 min. 10 min at 100 °C, stored on ice
repetition of in-vitro-cleavage assay SCL/TNT(+) with Ax7
25 µl TNT+, 4 µl buffer, 4 µl H2O, ad 40 µl H2O
4 µl Scl, 4 µl buffer, 25 µl H2O, ad 40 µl H2O
37 °C
06-01-16 Bianca SDS gel 15 µl probes for westernblot load on SDS gel (probes from 0, 30, 90 and 180 min + and -),40 min at 200 V
westernblot 90 min at 120 mA
membran swing for 1 h in milkpowder and TBST, 1 x in TBST washed, 1 x 5 min in TBST washed, 1 x 10 min in TBST washed
membran with TBST and α-His (antibody) over night (15 µl antibody in TBST (100 µg/500 µl))
06-02-16 Bianca, Janis membran washed, second antibody
06-03-16 Janis transformation chemically competent E.Coli cells were used, for Ax7L, Ax7R, scAx7L, 1-2; 2 µl DNA used and 50 µl competent cells; 20 min on ice, 45 sec at 42 °C, 1 h at 37 °C in 1 ml LB without antibiotic.
cells with Ax7L, Ax7R, scAx7L plated on LB and ampicillin. Cells with 1-2 on LB with chloramphenicol. Stored at 37 °C over night.
06-07-16 Wiebke, Theresa, Sabine Interlab study vector transformed into Top10 E.coli via heatshock transfomation. 1 h at 37 °C
06-27-16 Bianca assembly of TALENs new vector: p3H001; 4 rudiments with 1) Ax7L-DS, 2) Ax7R-RR, 3) Ax7L-scFOK, 4) Ax7R-scFOK
p3H001 77,0 ng/µl, 2 µl of C-terminal and 2 µl of N-terminal module, 5 µl H2O, used PCR programm (see protocol)
transformation transformation into E.coli with electroporation. 200 µl LB and 30 min at 37 °C. Plated on LB chloramphenicol, stored at 37 °C over night.
06-28-16 Bianca overnight cultures 5 ml LB and 34 mg/ml chloramphenicol with 1 colony from the plates. 3 cultures picked from each plate. Incubate at 37 °C over night.
06-30-16 Bianca, Kim plasmid isolation plasmid isolation with QIAprep Spin Miniprep kit. The protocol attached to the kit was used without step 7. Stored at -20 °C
overnight cultures 3 colonies from each plate picked, overnight cultures with 5 ml LB, 34 mg/ml chloramphenicol.
07-01-16 Kim control digest control digest with the isolated plasmids from 06-30-16. 10 µl plasmid DNA, 2 µl 10x reaction buffer, 0,5 µl EcoRI, 0,5 µl PstI
1 h at 37 °C and 10 min at 65 °C
agarose gel loaded with 15 µl probes from control digest and 7 µl 1 kb plus DNA ladder
plasmid isolation used the overnightcultures from 06-30-16; with QIA Prep Spin Miniprep Kit (without step 7)
07-19-16 Janis retransformation plasmids 1.1, 2.1, 3.1, 3.2, 4.1 in Top10 E.coli. Incubation in LB with chloramphenicol (1.1: Ax7L-DS, 2.1: Ax7R-RR, 3.1: Ax7L-scFOK, 3.2: Ax7L-scFOK, 4.1: Ax7R-scFOK)
07-20-16 Kim plasmid isolation used the cultures from 07-19-16, with QIA Prep Spin Miniprep Kit (without step 7)
07-21-16 Janis control digest used plasmids from 06-20-16, with 4 µl plasmid DNA, 0,5 µl XbaI and 0,5 µl PstI, 2 µl buffer and 10 µl H2O. For 1 h at 37 °C and 10 min at 65 °C.
07-28-16 Sabine, Wiebke, Theresa PCR run a PCR with 10 µl 5x HF Phusion Buffer, 5 µl dNTPs, 2,5 µl forward Primer, 2,5 µl reverse Primer, 1 µl template TCR5 H2, 0,5 µl Phusion polymerase, 28,5 µl H2O
1. programm: 98 °C 2 min at the beginnig, denaturation at 98 °C for 10 sec, annealing at 60 °C for 10 sec, extension at 72 °C for 30 sec. steps repeated for 35 times. at the and 72 °C for 5 min.
2. programm: same as the first programm, just the annealing temperature was 66 °C.
agarose gel probes loaded on 1 % agarose gel, run for 120 V for 20 min, 1 kb plus DNA ladder.
transformation 1.1, 2.1, 3.1, 3.2 and 4.1 in BL21DE3 E.coli with electroporation. 1 h at 37 °C , plated on LB with chloramphenicol, incubation at 37 °C
puc57 in Top10 with heatshock at 42 °C for 40 sec, 200 µl LB added, 1 h at 37 °C. Plated on LB
gel elution cut the fragment out of the gel. add 300 µl QG buffer, 10 min at 50 °C, add 100 µl Isopropanol, transfered to column, 1 min at max. rpm, add 400 µl QG buffer, 1 min at max. rpm, add 700 µl PE buffer,
1 min at 1500 rpm, centrifuge to dry the column, 1 min at max rpm, add 15 µl EB buffer or H2O, incubate for one minute, centrifugation for 1 min at max rpm.
cut and ligation 2 µl cut smart buffer, 2 µl ATP, 2 µl puc57, 1 µl SmaI, 1 µl T4 Ligase, 15 µl Elution, 2 µl H2O, for 2 h at 30 °C
PCR repeat the PCR, but used 1 min elonation time at 72 °C
agarose gel load gel with PCR probes, run at 120 V for 20 min
08-01-16 Wiebke, Sabine Plasmid isolation Plasmid isolation from overnight cultures with iGem Nterm and GFP puc57 (1, 2, 3, 4)
Used QIA Prep Spin Miniprep Kit without step 7. 4 ml Overnightculture were used.
control digest with the isolated plasmids. used 0,5 µl BsaI, 7 µl DNA, 2 µl CutSmart buffer, ad 20 µl (10.5 µl H2O)
1 h at 37 °C, 10 min at 65 °C.
agarose gel 1 % agarose gel, used 7 µl 1 kb DNA ladder, 20 µl from Controll digest with 6 µl 5x LD buffer (20 µl loaded on agarose gel)
Gel at 120 V for 25 min
08-02-16 Wiebke, Sabine, Louise plasmid isolation Repeat the plasmid isolation from 08-01-16
control digest with the isolated plasmids. used 1 µl BsaI, 26 µl DNA, 3 µl CutSmart buffer
agarose gel 1 % agarose gel, used 7 µl 1 kb DNA ladder, 20 µl from control digest with 6 µl 5x LD buffer (20 µl loaded on agarose gel)
08-03-16 Janis
cloning GFP - NTH3 Fragment into puc57: 5 µl DNA, 1 µl SmaI, 1 µl T4 Ligase, 1 µl Puc57, 1 µl ATP, 1 µl tango buffer; 1 h at room temperatur (RT)
transformation puc57 with GFP - NTH3 in E.coli (Top10)
transformation transformation into E.coli (BL21DE3) with electroporation. 200 µl LB and 30 min at 37 °C. Plated on LB chloramphenicol, stored at 37 °C over night.
Used the DNA 1.1, 2.1, 3.1, 3.2 and 4.1
08-10-16 Kim overnightcultures used 4 colonies from 08-03-16 (puc57 with GFP-NTH3 in Top10), 5 ml LB, 5 µl carbenicillin.
08-11-16 Kim plasmid isolation plasmid isolation with the overnightcultures from 08-10-16, used 4 ml overnightculture and QIA Prep Spin Miniprep Kit without step 7.
08-12-16 Kim control digest plasmids from 08-11-16; 5 µl DNA, 2 µl CutSmart buffer, 0.5 BsaI, 12.5 µl E.coli ; 1 h at 37 °C, 10 min at 65 °C
agarose gel 1 % agarose gel loaded with 20 µl probe (20 µl control digest and 4 µl 6x loading buffer), 7 µl 1 kb DNA ladder; 27 min at 100 V
08-16-16 Theresa, Lousie, Severin PCR like the PCR from 07-28-16
agarose gel 1 % agarose gel loaded with 60 µl probe (50 µl PCR probe and 12,5 5x loading buffer), 8 µl DNA ladder; 20 min at 120 V
gel extraction gel extraction with MiniElute Extraction Kit
cloning like the cloning from 08-03-16, but CurSmart buffer was used
transformation 10 µl cloning stuff, 50 µl competent E.coli cells; add 200 µl LB for 30 min
10 µl puc57, 50 µl competent E.coli cells; add 200 µl LB for 30 min
08-17-16 Janis overnightcultures 5 colonies (1-5) picked from transformation (08-16-16), in 5 ml LB with 5 µl Carbenicellin
08-18-16 Janis plasmid isolation plasmid isolation (overnight cultures used from 08-17-16) with QIA Prep Spin Miniprep Kit without step 7.
control digest control digest with the isolated plasmids (06-18-16), same control digest like the digest from 08-12-16 (5 µl DNA, 2 µl CutSmart buffer, 0.5 BsaI, 12.5 µl H2O; 1 h at 37 °C, 10 min at 65 °C)
assembly assembly oft GFP-NTH3 into iGem vector; 2 µl iGem vector (50 ng/µl), 1 µl hexa-repeat LA, 1 µl hexa-repeat AB, 1 µl hexa-repeat BR (each 50 ng/µl), 2 µl N-terminal module (100 ng/µl), 2 µl C-terminal module (100 ng/µl), 2 µl NEB buffer,
1 µl enzym Mix (2 µl ATP, 2 µl NEB CutSmart, 1 µl T4-DNA Ligase, 1 µl BsaI) and 7 µl H2O
assembly program: 10 min at 40 °C, 10 min at 16 °C (3 cycles), 20 min at 50 °C, 20 min at 80 °C (Final)
transformation 15 µl assembly, 50 µl competent cells, transformation with electroporation, 200 µl LB, 1 h at 37 °C, plated on LB with chloramphenicol
08-22-16 Kim overnightcultures picked 6 colonies from 08-18-16, with LB and Chloramphenicol
08-23-16 Kim plasmid isolation plasmid isolation (used 4 ml overnightcultures from 08-22-16) with QIA Prep Spin Miniprep Kit without step 7
control digest with plasmids (08-23-16), used 7 µl DNA, 2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 10 µl H2O
1 h at 37 °C, 10 min at 65 °C
agarose gel 1 % agarose gel, loaded with 20 µl probes (20 µl control digest and 4 µl 6x loading buffer), 29 min at 100 V
08-24-16 Janis, Kim transformation plasmids 1.1, 2.1, 3.1, 3.2, 4.1 in BL21 E.coli., incubation on LB with Chloramphenicol at 37 °C
control digest repeat control digest from 08-23-16 with 10 µl DNA (2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 7 µl H2O); 1 h at 37 °C, 10 min at 65 °C
agarose gel 1 % agarose gel, loaded with 20 µl probes (20 µl control digest and 4 µl 6x loading buffer), 29 min at 100 V
overnightculures picked 4 colonies from 08-18-16, with LB and Chloramphenicol
08-25-16 Janis, Kim plasmid isolation plasmid isolation (overnightcultures used from 08-24-16 7-10(GFP-NTH3 in iGem vector)) with QIA Prep Spin Miniprep Kit without step 7.
plasmid isolation (overnightcultures interlab study + control 1, + control 2, - control 1, - control 2)
transformation retransformation plasmids 1.1, 2.1, 3.1, 3.2 and 4.1 in Top 10 E.coli, 50 µl competent cells, 20 min on ice, 45 sec at 42 °C, 1 h at 37 °C in 200 µl LB, plated on LB with chloramphenicol
transformation plasmids for interlab study in Top10 E.coli, 50 µl competent cells, 20 min on ice, 45 sec at 42 °C, 1h at 37 °C in 200 µl LB, plated on LB
08-26-16 Kim control digest with plasmids (25-08-16), used 11 µl DNA, 2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 6 µl H2O - different XbaI and PstI stocks used, also repeat control digest with plasmids 1-6
1 h at 37 °C, 10 min at 65 °C
agarose gel 1 % agarose gel, loaded with 20 µl probes (20 µl control digest and 4 µl 6x loading buffer), 29 min at 100 V
08-29-16 Bianca Interlab study, transformation DNA from Göttingen in 10 µl H2O; 50 µl competent cells (Top10 E.coli), 2 µl DNA, 20 min on ice, 45 sec at 42 °C, 1 ml LB 1 h at 37 °C, plated on LB with chloramphenicol, incubation at 37 °C
overnightcultures 3 colonies each plate (from 08-25-16, Top with 1.1, 2.1, 3.1, 4.1), with 5 ml LB and Chloramphenicol, incubate at 37 °C
3 colonies each plate (from 08-24-16, BL21 with 1.1, 2.1, 3.1, 3.2, 4.1) with 5 ml LB and Chloramphenicol, incubate at 37 °C
08-30-16 Bianca, Theresa expression 2.5 ml from each overnightculture (from 08-29-16, BL21 with 1.1, 2.1, 3.1, 3.2, 4.1) in 10 ml LB
Incubation, optical density after 60 min: 1.1 - 0.136, 2.1 - 0.122, 3.1 - 0.181, 3.2 - 0.153, 4.1 - 0.134
optical density after 115 min: 1.1 - 0.489, 2.1 - 0.494, 3.1 - 0.596, 3.2 - 0.520, 4.1 - 0.420
optical density after 150 min: 1.1 - 0.728, 2.1 - 0.540, 3.1 - 0.921, 3.2 - 0.792, 4.1 - 0.660
1 ml, centrifuged, pellet stored at -20 °C (pre induction)
add 238 µl IPTG, incubate for 1 h and 40 min at 37 °C, 1 ml centrifuged, pellet stored at -20 °C (after induction)
rest centrifuged, into liquid nitrogen and stored at -80 °C
plasmid isolation plasmid isolation (overnightcultures re-transformation from 08-29-16) with QIA Prep Spin Miniprep Kit without step 7
overnightcultures 100 µl overnightculture (from 08-26-16 interlab study) with 8 ml LB and chloramphenicol, incubation at 37 °C
08-31-16 Bianca, Theresa, Janis assembly with 1 µl GFP-NTH3, 1 µl vector (50 ng/µl), 1 µl HexaI (Repeats 1-6), 1 µl HexaII (Repeats 7-11,5), 1 µl T4-Ligase, 1 µl BsaI, 2 µl 10mM ATP, 2 µl CutSmart buffer, ad 20 µl H2O
two different vectors were used: iGem vector (with c-terminus c-63) and psaE6 (with c-terminus c-17) and 4 different Hexa combinations each vector
A: iGem vector and Hexa 42 + HDHx
B: iGem vector and Hexa 42 + 2xNG
C: iGem vector and Hexa 42 + 2xNN
D: iGem vector and pJet Hexa3 rep
E: psaE6 and Hexa 42 + HDHx
F: psaE6 and Hexa 42 + 2xNG
G: psaE6 and Hexa 42 + 2xNN
H: psaE6 and pJet Hexa3 rep
interlab study culture and measurment
09-01-16 Janis, Theresa transformation 15 µl assambly product, 50 µl competent cells (E.coli, Top10), 20 min on ice, 45 sec at 42 °C, add 200 µl LB, 1 h at 37 °C, plated on LB with Chloramphenicol
SDS gel prepared 4 gels
control digest with plasmids (08-25-16re-transformation), used 11 µl DNA, 2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 6 µl H2O
1 h at 37 °C, 10 min at 65 °C
09-05-16 Theresa, Kim SDS gel prepared 8 gels
separating gel (10 %): 27.25 ml H2O, 33.25 Acrylamid (30 %), 25 ml Tris (1.5 M; pH 8.8), 1 ml APS (10 %), 1 ml SDS (10 %), 40 µl TEMED
stacking gel: 34 ml H2O, 8.5 ml Acrylamid (30 %), 6.25 ml Tris (0.5 M; pH 6.8), 0.5 ml APS (10 %), 0.5 ml SDS (10 %), 50 µl TEMED
first separating gel, than stacking gel; ~1 ml Isopropanol on separating gel, when it is in the chamber (remove it before filling stacking gel in the chamber)
1. H2O, 2. Tris, 3. SDS, 4. Acrylamid, 5. APS, 6. TEMED; APS and TEMED everytime at last! After adding them be fast
overnightcultures with colonies from tranformation 09-01-16, 4 colonies of the plates A-D, there was nothing on the plates E-H; 5 ml LB, 5 µl Chloramphenicol and the picked colonie
09-06-16 Theresa, Kim plasmid isolation plasmid isolation (overnightcultures from 09-05-16, A1-3, B1-3, C1-3, D1-3) with QIA Prep Spin Miniprep Kit without step 7
transformation used plasmids from assembly F, G and H (08-31-16), 10 µl assambly product, 50 µl competent cells (E.coli, Top10), 20 min on ice, 45 sec at 42 °C, add 200 µl LB, 30 min at 37 °C, plated on LB with Chloramphenicol
09-07-16 Theresa, Bianca, Kim control digest with plasmids (08-25-16), used 4 µl DNA, 2 µl 10x reaction buffer, 0.5 µl XbaI, 0.5 µl PstI, 13 µl H2O
1 h at 37 °C, 10 min at 65 °C
agarose gel 1 % agarose gel, loaded with 20 µl probes (20 µl control digest and 4 µl 6x loading buffer), 29 min at 100 V
SDS gel prepared probes: took a little bit of the pellets (from the expression on 08-30-16), also the pre-induction and the after-induction pellets; 80 µl Leammli, resuspent, 10 min at 100 °C
load gel with 15 µl of the prepared probes, 5 µl Page Ruler best; run for 42 min; gel in coomassi staining solution
transformation 15 µl plasmid DNA (from plasmid isolation 09-06-16, plasmids A3, A4, B1, B2, C2, D1, D2) 50 µl competent cells (E.coli, BL21), 20 min on ice, 45 sec at 42 °C, add 200 µl LB, 35 min at 37 °C, 100 µl plated on LB with Chloramphenicol
09-08-16 Theresa, Kim SDS gel gel 3 h in H2O and 4 h in destaining solution, after that over night in PBS buffer
transformation repeat transformation from 08-07-16, nothing changed
09-09-16 Kim overnightcultures used plates from transformation (09-07-16), each plate (A3, A4, B1, B2, D2) one colonie picked, 5 ml LB and 5 µl Chloramphenicol (34 mg/ml)
09-12-16 Kim overnightcultures used plates from transformation (09-07-16), each plate (A3, A4, B1, B2, C2, D2) one colonie picked, 5 ml LB and 5 µl Chloramphenicol (34 mg/ml)
09-13-16 Theresa, Sabine, Wiebke Expression Used the overnightcultures from 09-12-16, 100 ml LB and different amounts of overnightcultures, depents on the optical density. Used 1.17 ml A, 1.8 ml B, 3.22 ml C, 2.4 ml D - Start optical density was 0.05
Incubation; optical density after 1 h: A - 0.063, B - 0.11, C - 0.201, D - 0.115
Optical density after 2.5 h: A - 0.452, B - 0.59, C - 0.93, D - 0.756
Optical density after 2.75 h: A - 0.5
Optical density after 3 h: A - 0.59
1 ml, centrifuged, pellet stored at -20 °C (pre induction)
add 200 mM IPTG (1 ml), incubate for 1 h and 30 min at 37 °C, 1 ml centrifuged, pellet stored at -20 °C (after induction)
rest centrifuged 10 min, into liquid nitrogen and stored at -80 °C
09-14-16 Sabine, Wiebke, Louise, Theresa, Kim, Severin Purification of tales Resuspensionof sample 1.1.1 in 2ml TE-Buffer and 20 μl PMSF
Ultrasonic treatment on ice: program9, 30%energy for 8 min (30sec on/30 sec off)
Centrifuged at 4°C, 20.000gfor 15 min
Supernatant transferred to column(Gravity flow Strep Tactin PepharoseColumn, Iba), purification as
stated by iba protocol
Flow through was stored as aliquots of 500µl labelled ‘E 1 – 6’ and ‘W 1 – 5’. Flow through of first centrifugation was stored and labelled as ‘supernatant’. Storage at -20°C.
09-15-16 Theresa, Wiebke, Kim, Sabine, Severin SDS-Gels with samples derived from TALe 1.1.1 (09-14-2016) 50µl of each sample mixed with 5,88µl TCA (final concentration 10%). Incubated for 10 min on ice. Centrifuged at 4°C, 20 000xg for 10 min. Mixed with 500µl of 80% acetone and centrifuged at 4°C, 20 000xg for 10 min.
Incubated for 30 min at 37°C. Mixed with 40µl Laemmli and incubated for 10 min at 80°C. Gels ran for 1 h and 20 min at 200V in TANK buffer.
Coomassie-Staining Coomassie-gels were set to stain over night.
Western Blot Other gels were Blotted semi-dry at 70V for 50 min. Poncau-staining was done for 10 min. Incubated for 1 h in PBST with 3% milkpowder. Incubation over night with 1st antibody
(StrepMAB-Classic Cell Culture Supernatant Iga1 anti-strep-tag II monoclonal, 2-1508-025 Iba life science)
09-16-16 Sabine, Kim, Wiebke Continuation of Western 1st antibody removed by washing in PBST. Incubation in PBST with 2nd antibody (Ecl anti-mouse IgG horseradish peroxidase) for 3 h
Stained with 6 ml 100 mM TRIS (pH 8,5), 30 µl Luminol (22.2 mg/ 500 µl DMSO), 15 µl p-Coumaric acid (7.4 mg/ 500 µl DMSO) and 10 µl 30% H2O2 for 1 min up to 30 min
Continuation of Coomassie-Staining Destained for 1,5 h and incubated in water for 1h
09-20-16 Sabine, Wiebke, Kim, Theresa, Bianca, Louise Purification of tales Resuspension of sample 1.1.1, B2.1 and C2.1 in 2 ml TE-Buffer and 20 µl PMSF, Ultrasonic treatment on ice: program 9, 30% energy for 8 min (30 sec on/30 sec off). Centrifugation at 4°C, 20 000xg for 15 min
Supernatant transferred to column (Gravity flow Strep Tactin Pepharose Colum, Iba), purification as stated by Iba protocol
Flow through was labelled as ‘E 1 – 6’ and ‘W 1 – 5’ and stored at -20°C
Others Fluorecence of GFP-TAL fusion proteins in BL21 strains 1.1.1, B1 and C2 and in corresponding raw extracts was documented
expression Overnight cultures in 200 ml LB of BL21 strains B2 and C2 (09-12-16) inoculated and incubated at 37°C
overnight cultures Overnight cultures in 5ml LB and master dish of 2.1.1, 3.1.1, 3.2.1 and 4.1.1 (09-12-16) inoculated and incubated at 37°C
09-21-16 Bianca TEV digestion According to ProTEV plus protocol (Promega)
2µl Buffer
0,4 µl 100mM DTT
36,8 µl Protein (E6,5) as collected on 09-14-16
0,8µl TEV
Incubated at 30°C. Samples of 9,5µl were taken after 0, 1, 2, 4 and 6h and stored at -20°C
Proteinexpression Overnight cultures of BL21 strains B2 and C2 (09-20-16) were used to inoculate 5x 200ml LB+CA each and grown at 37°C to an OD600 of approx. 0,500.
1ml samples were taken of each flask, the cells pelleted, labelled as ‘before induction’ and stored at -20°C.
Protein expression was induced with 400 µl IPTG, final concentration: 2mM
After 1,5h 1ml samples were taken of each flask, the cells pelleted, labelled as ‘after induction’ and stored at -20°C
and the remaining cells were harvested and stored at -80°C
TCA precipitation Samples derived from 1.1.1, B2.1 and C2.1 (09-20-16) were mixed with 3 µl NaDOC and incubated for 10min on ice
3 µl TCA were added and samples were incubated for 60 min on ice, Centrifuged at 4°C, 6 500RPM for 20 min, Pellets were resuspended in 1 ml 80% acetone,
Centrifuged at 4 °C, 6,500RPM for 10 min, Supernatant was discarded; samples were dried at 37 °C, 30 µl SDS running-buffer + Lämmli was added, incubated at 80 °C for 10 min, Samples were stored at -20 °C
other Overnight culture of 1.1.1 was transferred into 30ml fresh LB+CA and incubated over night at 37 °C
09-22-16 Bianca, Janis Proteinexpression Overnight culture of 1.1.1 (09-21-16) was used to inoculate 5x 200ml LB+CA each and grown at 37 °C to an OD600 of approx. 0,600. 1 ml samples were taken of each flask, the cells pelleted, labelled as ‘before induction’ and stored at -20 °C.
Proteinexpression was induced with 400 µl IPTG, final concentration: 2 mM
After 1,5h 1 ml samples were taken of each flask, the cells pelleted, labelled as ‘after induction’ and stored at -20 °C and the remaining cells were harvested and stored at -80 °C
SDS-gels Gels ran for 40 min at 200V in TANK buffer
Western Blot semi-dry blot at 6,9 V for 45 min
Poncau-staining was done for 10 min
Incubation for 1 h in PBST with 3 % milkpowder
Incubated at 4°C over night with 1st antibody (StrepMAB-Classic Cell Culture Supernatant Iga1 anti-strep-tag II monoclonal, 2-1508-025 Iba life science
Plasmidisolation, With Quigen Mini Prep Spin Column Kit according to protocol.
Masterdish Petridish was damaged, redone with recent BL21 strains: A3.1, A3.2, A3.3, A4.1, A4.2, A4.3, B1.1, B1.2, B1.3, B2.1, C2.1, C2.2, C2.3, D1.1
09-23-16 Bianca, Janis, Kim Control digest Plasmids isolated on 09-22-16 were digested with PstI and XbaI (Set1) as well as PstI, XbaI and BsaI (Set2)
Continuation of Western Blot 1st antibody removed by washing in PBS, Inbubation with 2nd, Incubated in PBST with 2nd antibody (EcL anti-mouse IgG horseradish peroxidase) for 2h, 2nd antibody removed by washing in PBS,
Stained with 6 ml 100mM TRIS (pH 8,5), 30 µl Luminol (22,2 ng/500µl DMSO), 15 µl p-Coumaric acid (7,4 ng/500 µl DMSO) and 10 µl 30% H2O2 for 1 min up to 30 min
09-26-16 Kim, Bianca SDS-Gel Used premade 10% Gel from Biorad, TEV-digestion (09-21-16) samples were mixed with 10 µl Lämmli each, E6 1.1.1, B, C (09-20-16) samples were mixed with 30 µl Lämmli each, Incubation at 100°C for 10m in
Gels ran for 45 min at 150 V in TANK buffer.
Western Blot Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) Blotted at 6V for 40min in fastBlot Trans Blot Turbo
Poncau-staining was done for 10 min
Incubated for 1h in blocking solution (1xPBST with 1xRotiBlock, Roth), washed with PBST
Incubated at 4°C over night with 1st antibody
Coomassie staining Coomassie-gels were set to stain over night
Overnight cultures 3.1.1 and 4.1.1 were inoculated in 25ml LB+CA and grown overnight at 37°C
09-27-16 Bianca, Kim, Wiebke TEV digestion According to ProTEV plus protocol (Promega)
2µl buffer, 0,4 µl DTT, 36,8µl sample (1.1.1 E6 as collected on 09-20-16), 0,8 µl TEV
Incubated at 30 °C. Samples of 9,5 µl were taken after 0, 1, 2, 4 and 6 h and stored at -20 °C
continuation of Coomassie-Staining After incubation with destaining solution no bands, not even marker, were visible. Gel discarded.
continuation of Western Blot 1st antibody removed by washing in PBS
Incubated in PBST with 2nd antibody (EcL anti-mouse IgG horseradish peroxidase) for 1 h
2nd antibody removed by washing in PBS
Stained with 6 ml 100 mM TRIS (pH 8,5), 30 µl Luminol (22,2 ng/500 µl DMSO), 15 µl p-Coumaric acid (7,4 ng/500 µl DMSO) and 10µl 30% H2Osub>2
Proteinexpression Overnight cultures of BL21 3.1.1 and BL21 4.1.1 (09-26-16) was used to inoculate 50 ml LB+CA each and grown at 37 °C to an OD600 of approx. 0,600.
1ml samples were taken of each flask, the cells pelleted, labelled as ‘before induction’ and stored at -20 °C.
Proteinexpression was induced with 100 µl IPTG, final concentration: 2mM
After 1,5 h 1 ml samples were taken of each flask, the cells pelleted, labelled as ‘after induction’ and stored at -20 °C and the remaining cells were harvested and stored at -80 °C
09-28-16 Kim, Bianca Purification of tales Resuspension of samples 1.1.1 (09-22-16), B and C (09-21-16) as well as 3.1.1 and 4.1.1 (09-27-16) in 2 ml TE-Buffer and 20 µl PMSF, Ultrasonic treatment on ice: program 9, 30% energy for 8 min (30 sec on/30 sec off)
Centrifuged at 4 °C, 20 000xg for 15 min
Supernatant transferred to column (Gravity flow Strep Tactin Pepharose Colum, Iba), purification as stated by Iba protocol
09-29-16 Janis, Bianca, Kim SDS-Gel Proben: Raw extract B from 09-21-16, E6 B from 09-20-16, E6 B from 09-28-16
Raw extract C from 09-21-16, E6 C from 09-20-16, E6 C from 09-28-16
Raw extract 1.1.1 from 09-22-16, E6 1.1.1 from 09-22-16, E6 1.1.1 from 09-28-16
Raw extract 3.1.1, E6 3.1.1 from 09-28-16
raw extract 4.1.1, E6 4.1.1 from 09-28-16
TEV 0 from 09-27-16, TEV 1 from 09-27-16, TEV 2 from 09-27-16, TEV 4 from 09-27-16
Coomassie-staining Incubation overnight at RT
Western Blot Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)), Blotted at 6V for 40 min in fastBlot Trans Blot Turbo
Poncau-staining was done for 10min
Incubated for 1h in PBST with 3% milk powder, washed with PBST
09-30-16 Kim Continuation of Coomassie-Staining Destained Gels
continuation of Western Blot Washed membrane with PBS
Incubated with 1st antibody (StrepMAB-Classic Cell Culture Supernatant Iga1 anti-strep-tag II monoclonal, 2-1508-025 Iba life science) for 1 h
1stAntibody removed by washing in PBS
Inbubation with 2nd antibody (EcL anti-mouse IgG horseradish peroxidase) for one hour
2nd Antibody removed by washing in PBS
Stained for 1 min with 6 ml 100 mM TRIS (pH 8,5), 30 µl Luminol (22,2 ng/500 µl DMSO), 15 µl p-Coumaric acid (7,4 ng/500 µl DMSO) and 10 µl 30% H2O2
010-04-16 Kim, Severin, Wiebke Colonie-PCR Single Colonies of B, C and 1.1.1 were suspended in 10 µl H2O and incubated at 95°C for 5min.
1µl sample and 49µl mastermix were prepared and amplified as follows:
50µl Buffer 5xG
5µl dNTPs
10µl Primer 1 (1534)
10µl Primer 2 (1716)
7.5µl DMSO
135µ l H2O
25µl MgCl2
Program:
35x (30s 98°C, 30 s 59 °C, 30 s 72 °C) and 5 min 72 °C
agarose gel 1 % agarose Gel, Gels ran for 27min at 100V
Proteinexpression 1ml LB+CA was inoculated with strain C as a preculture and later distributed into 10x 10ml LB.
Cultures were grown to an OD600 of approx. 0,5 as well as 0.847
Proteinexpression was induced at 30°C and 37°C with 2mM IPTG for as long as 3h and over night.
10-05-16 Kim, Severin, Wiebke Liquid cultures were harvested and split into two separate reaction tubes for Dotplot and Immunostain
10-06-16 Kim, Wiebke TEV digestion Using samples according to ProTEV plus protocol (Promega)
5µl Buffer
1 µl 100 mM DTT
40 µl Sample 1.1.1 (09-08-16) and C (09-20-16, 20 µl E5 and 20 µl E6)
2 µl TEV
52 µl H2O
Incubated at RT. Samples of 20 µl were taken after 0, 1, 2, 4 and 6h and stored at -20°C
SDS-Gel 20µl of TEV digestion sample was mixed with 20µl Lämmli and incubated at 80°C for 10min, Gels (10%) ran for 50min at 150V in TANK buffer.
Western Blot Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3))
Blotted at 25V for 40min in fastBlot Trans Blot Turbo
Poncau-staining was done for 10min
Washed with H2O
Incubated for 1h in blocking solution (1x PBST with 1x RotiBlock, Roth)
Washed with PBS
Incubated at 4°C over night with 1st antibody
10-07-16 Wiebke, Severin, Kim Continue the Immunostain. Wash the Membran with PBS
Incubate at RT with the second antibody for one hour
Wash with PBS
Stained for 1min with 6 ml 100 mM TRIS (pH 8.5), 30 µl Luminol (22.2 ng/500 µl DMSO), 15 µl p-Coumaric acid (7.4 ng/500 µl DMSO) and 10 µl 30% H2O2
Spotting the Oligonucleotides on a Chip iGEM.npw as layout
Solutions on Lafontain plates, 10 µl Oligonucleotides (100 µM) + 10 µl array it Micro spotting (2x)
spotting
Aftertreatment:
1) UV-Crosslink for 3 minutes
2) 24 hours in a dark room at room temperature (RT)
3) reducing: 2 min 0.2 % SDS, 2x 1 min H2O, 5 min NaBH4, 1 min icecold H2O, 1 min 0.2 % SDS, 2x 1 min H2O
dry
4) Blocking: 45 min Blocking reagent, 42 °C 5 min H2O
dry
Overnightcultures Used glycerin stock from Origami cells, incubate in 20 ml LB medium at RT over the weekend
Used colonies from 1.1.1 and 2.1.1 from masterdish, 50 ml LB medium and 50 µl Chloramphenicol. Incubate over the weekend at RT
10-10-16 Kim, Wiebke, Severin Purification Samples of 1.1.1 (09-20-16) and 2.1.1 (08-30-16) were resuspended in 5ml TE-Buffer with 50 µl PMSF. Proteins were purifies according to Iba protocol
One Column of 2.1.1 was incubated over night with 50 mM DTT. (at the 5th washing step, use 2 column bead volume (0.4 ml) and add 50 mM DTT. Incubate over night)
Proteinexpression Cultures of 1.1.1 and 2.1.1 were inoculated into 5x 200ml LB+CA
Cultures were grown to an OD600 of approx. 0,5-0,6
Proteinexpression was induced at 30°C and 37°C with 2mM IPTG for as long as 1,5h.
Afterwards cells were harvested and stored at -80°C
Prepation of electro competent cells Take 1 ml or more from the overnight culture from origami cells into 50 ml LB medium. Grow the cells until they reaches a optical density at 600 nm of 0.5 – 0.6. Incubate the bacteria 20 min on ice.
Centrifuge 10 min at 4 °C and discard the supernatant. Add 20 ml cold H2O and resuspend the pellet. Centrifuge again 10 min at 4 °C.
Add 20 ml cold H2O and centrifuge for 10 min at 4 °C. Discard the supernatant and add 20 ml cold H2O. Centrifuge for 10 min at 4 °C. Discard the supernatant and add 10 ml cold glycerin (10 %).
Resuspend the pellet and centrifuge at 4 °C for 10 min. discard the supernatant and resuspend the pellet in 2 ml 10 % cold glycerin
Take 100 µl aliquots into 1.5 µl reaction tubes. Froze them in liquid nitrogen and store at - 80 °C or use them directly for transformation.
transformation electropotation of competent origami cells with 1 µl plasmid DNA (from 1.1.1 and 2.1.1)
incubate for 30 min in 500 µl LB at 37 °C. Plate 100 µl on LB with Chloramphenicol. incubate at 37 °C over night
10-11-16 Wiebke, Louise, Kim Continued Purification More samples of 1.1.1 (09-20-16) and 2.1.1 (08-30-16) were resuspended in 5 ml TE-Buffer with 50 µl PMSF. Proteins were purifies according to Iba protocol
One Column of 1.1.1 and 2.1.1 were incubated overnight with 50 mM DTT.
the incubate column from 10-10-16 were Eluated
Stability test of circularised TALes Purified protein samples of 2.1.1 were set to incubate with DTT (circularised) or without (control) for 0, 6, 24, 32, 48 h at 4 °C, RT and 37 °C.
In addition, one set of samples was frozen at –20°C and thawed at RT for 30 min each for four continuous times.
The linearized TAL 3HD100 was used as an additional control. After each time a fraction of 10µl was stored at -20°C for analysis.
SDS-Gel 10µl of sample were mixed with 10µl Lämmli and incubated at 80°C for 10min.
10µl of the sample/Lämmli mixture were put onto the Gels (10%) which ran for 50min at 150V in TANK buffer.
western blot Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3))
Blotted at 25V for 40min in fastBlot Trans Blot Turbo
Incubated for 1h in blocking solution (1x PBST with 1x RotiBlock, Roth)
Washed with PBS
Incubated at 4°C over night with 1st antibody
Overnightculture Used colonies from 1.1.1 and 2.1.1 fromtransformation with origami, 20 ml LB medium and 20 µl Chloramphenicol. Incubate at 37 °C over night
10-12-16 Wiebke, Louise, Sabine, Kim Proteinexpression Cultures of 1.1.1 and 2.1.1 were inoculated into 2x 125ml LB+CA
Cultures were grown to an OD600 of approx. 0,5-0,6
Proteinexpression was induced at 30 °C and 37 °C with 2 mM IPTG for as long as 2 h.
Afterwards cells were harvested and stored at -80°C
TEV digestion Using samples 1.1.1 +/- DTT (10-11-16) and 2.1.1 +/- DTT (11-10-16) according to ProTEV plus protocol (Promega)
Samples were taken after 0, 1, 2, 4 and 6h and stored at -20°C
Continue Immunostain wash membran with PBS
Incubate second antibody for 1 hour
wash with PBS
Stained for 1min with 6ml 100mM TRIS (pH 8.5), 30µl Luminol (22.2ng/500µl DMSO), 15µl p-Coumaric acid (7.4ng/500µl DMSO) and 10µl 30% H2O2
10-13-16 Wiebke, Kim, Sabine, Louise SDS-Gel TEV digested samples (10-12-16) were mixed 1:1 with Lämmli and incubated at 80°C for 10min.
Gels (10%) ran for 50min at 150V in TANK buffer.
Stability test of circularised TALes Purified protein samples (10-13-16), 1.1.1 (E5 + E6) and 2.1.1 (E5 + E6), were set to incubate for 0, 15 min, 5 h and 20 h at 4°C, RT and 37 °C.
In addition, one set of samples was frozen at –20 °C and thawed at RT for 30min each for four continuous times. Each time a fraction of 10 µl was stored at -20 °C for analysis.
Western Blot and Immunostain Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3))
Blotted at 25V for 40min in fastBlot Trans Blot Turbo
Incubated for 1h in blocking solution (1x PBST with 1x RotiBlock, Roth)
Washed with PBS
Incubated for 3h at RT with 1st antibody (2 times Flag antibody, to times Strep antibody at TEV digest Western blot)
10-14-16 Wiebke, Kim, Sabine Continue Immunostain 1stAntibody removed by washing in PBS
Inbubation with 2nd antibody
2nd Antibody removed by washing in PBS
Stained for 1 min with 6 ml 100mM TRIS (pH 8,5), 30µl Luminol (22,2 ng/500 µl DMSO), 15 µl p-Coumaric acid (7,4 ng/500 µl DMSO) and 10 µl 30% H2O2
Stability test of circularised TALes Purified protein samples (10-13-16 and 10-14-16) of Origami with DTT were set to incubate for 6 h, 48 h ... at 4 °C, RT and 37 °C.
In addition, one set of samples was frozen at –20 °C and thawed at RT for 30 min each for four continuous times. Each time a fraction of 10 µl was stored at -20 °C for analysis.
Using samples E1 – E6 from 10-13-16 according to ProTEV plus protocol (Promega)
Samples were taken after 0, 1, 2, 4 and 6h and stored at -20°C
10-16-16 Wiebke stability Test Probe at 4 pm (68 h for probes without DTT and 54 h for the probes with DTT)
10-17-16 Wiebke, Louise, Sabine, Theresa, Kim SDS-gels Used 8 SDS Gels for the probes from the two stability tests with and without DTT. One gel was used for the TEV digest.
Gels run for 50 min at 150 V.
Western blot Used Towbin-Buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3))
Blotted at 25 V for 40min in fastBlot Trans Blot Turbo
Incubated for 1h in blocking solution (1x PBST with 1x RotiBlock, Roth)
Washed with PBS
Immunostain Incubated Membrane 1-6 for 2 h at RT with 1st antibody and membrane 7-9 over night at 4 °C
1stAntibody removed by washing in PBS
Inbubation with 2nd antibody for 1 h
2nd Antibody removed by washing in PBS
Stained for 1 min with 6 ml 100mM TRIS (pH 8,5), 30 µl Luminol (22,2 ng/500 µl DMSO), 15 µl p-Coumaric acid (7,4 ng/500 µl DMSO) and 10 µl 30% H2O2/td>
10-18-16 Kim Continued immunostain from 10-17-16 1stAntibody removed by washing in PBS
Inbubation with 2nd antibody for 1 h
2nd antibody removed by washing in PBS
Stained for 1 min with 6 ml 100 mM TRIS (pH 8,5), 30 µl Luminol (22,2 ng/500 µl DMSO), 15 µl p-Coumaric acid (7,4 ng/500 µl DMSO) and 10 µl 30% H2O2

Protocols

Sponsors

Our project would not have been possible without financial support from multiple sponsors and supporters.
Carl Roth IDT Leibniz University Hannover Leibniz Universitätsgesellschaft e.V. New England Biolabs Promega Sartorius SnapGene

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