iGEM 2016 — Team Hannover

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# Team:Hannover/Notebook

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#### [**Notebook**](#collapseMenu)

#### [**Protocols**](#protocols)

# 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

* [Protocol TALE in BL21](https://static.igem.org/mediawiki/2016/7/7b/T--Hannover--Protocol-iGEM-TALE-in-BL21.pdf)
* [Protocol TALE in Origami](https://static.igem.org/mediawiki/2016/2/27/T--Hannover--Protocol-iGEM-TALE-in-Origami.pdf)
* [Protocol TALE with GFP](https://static.igem.org/mediawiki/2016/c/c2/T--Hannover--Protocol-iGEM-TALE-with-GFP.pdf)

![](https://static.igem.org/mediawiki/2016/7/72/T--Hannover--background.png)

##### **Sponsors**

**Our project would not have been possible without financial support from multiple sponsors and supporters.**

[![](https://static.igem.org/mediawiki/2016/b/bf/HQ_menu_logo.jpg)](https://2016.igem.org)