Team:Munich/Notebook

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Labbook We documented our work chronologically on a daily basis, linked the respective protocols and added all lab results. You can find the entries sorted according to month and sub-project, which can be selected separately. To do actual experiments it is also important to prepare buffer and all kind of stock solutions, agar plates, gels and so on. This basic work is partially not mentioned here, but was also done by the team. To ç To find what was done in each day, select the month you want to see and then select which category you want to check out. Cas13a Read-out Target Other Tuesday 25th Plating of DH5α p2CT-His-MBP-Lbu-Cas13a-WT and DH5α p2CT-His-MBP-Lsh-Cas13a-WT on LBCarb agar plates Protocol: - Participants: Ludwig Observations: Incubation at 37 °C over night. Results Bacteria grew and single colonies were visible. Wednesday 26th Preparation of 6 ml LBCarb cultures of DH5α p2CT-His-MBP-Lbu-Cas13a-WT and DH5α p2CT-His-MBP-Lsh-Cas13a-WT on LBCarb agar plates Protocol: - Participants: Ludwig Observations: Incubation at 37 °C over night. Results Bacteria cultures grew. Thursday 27th Preparation of cryo stocks for DH5α p2CT-His-MBP-Lbu-Cas13a-WT and DH5α p2CT-His-MBP-Lsh-Cas13a-WT Protocol: Cryo stocks Participants: Ludwig Minipreparation of DH5α Cas13a overnight cultures Protocol: Minipreparation Participants: Ludwig Electro transformation of p2CT-His-MBP-Lbu-Cas13a-WT and p2CT-His-MBP-Lsh-Cas13a-WT in E. coli Rosetta. Protocol: Electro transformation Participants: Ludwig Observations: 1 µl of plasmid DNA was used. Bacteria were plated on LBCarb plates without additional Cm. Results Bacteria lost second plasmid probably. Tuesday 2nd Electro transformation of p2CT-His-MBP-Lbu-Cas13a-WT and p2CT-His-MBP-Lsh-Cas13a-WT in E. coli Rosetta. Protocol: Electro transformation Participants: Ludwig Observations: 1 µl of plasmid DNA was used. Bacteria were plated on LBCarb + Cm plates Results Bacteria grew and single colonies were visible. Preparations of buffers for Cas13a purification. Protocol: Protein purification Participants: Chris, Ludwig, Julian Observations: TCEP changes the pH. Readjustement after adding neccessary. Electro transformation of p2CT-His-MBP-Lbu-Cas13a-WT and p2CT-His-MBP-Lsh-Cas13a-WT in E. coli Rosetta. Protocol: Electro transformation Participants: Ludwig Observations: 1 µl of plasmid DNA was used. Bacteria were plated on LBCarb + Cm plates Results Bacteria grew, but only few single colonies were visible. Wednesday 3rd Electro transformation of p2CT-His-MBP-Lbu-Cas13a-WT and p2CT-His-MBP-Lsh-Cas13a-WT in E. coli Rosetta. Protocol: Electro transformation Participants: Ludwig, Chris Observations: 1 µl of plasmid DNA was used. Bacteria were plated on LBCarb + Cm plates Results This time bacteria grew well and a lot of single colonies were visible. Preparation of 25 ml LBCarb + Cm cultures of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT and Rosetta p2CT-His-MBP-Lsh-Cas13a-WT and incubated at 37 °C overnight. Protocol: Protein Expression Participants: Ludwig Thursday 4th Harvesting of Rosetta cells with overexpressed Cas13a Lbu and Cas13a Lsh Protocol: Protein expression Participants: Ludwig, Chris Results Bacterial pellets were stored at -80 °C. Friday 5th Protein purification of Cas13a Lbu Protocol: Äkta protein purification Participants: Ludwig, Chris Observations: One test sample was run on the Äkta before the actual run. Only the second run was analyzed with a SDS PAGE. Results Protein purification of Cas13a Lsh Protocol: Äkta protein purification Participants: Ludwig, Chris Results Monday 8th Transformation of TEV plasmid (form lab in the chemistry department) in DH5α Protocol: Electro Transformation Participants: Sven Tuesday 9th Dialysis of pooled peak fractions of Cas13a Lbu and Cas13a Lsh into ion exchange buffer Protocol: Äkta protein purification Participants: Ludwig, Chris, Sven PCR of NT and T crDNA (for Cas13a Lbu and Cas13a Lsh) Protocol: Participants: Sven Observations: PCR didn't work. Wednesday 10th Test of different lysis methods for total RNA extraction with Norgen kit Protocol: Total RNA Purification with Norgen Kit Participants: Julian, Milica, Jorge Observations: Three lysis methods were used: sonifcation, lysozyme digestion and heat lysis in SDS. Lysozyme digestion: According to protocol. Heat lysis: Sample was incubated in 2% SDS at 75 °C for 15 min. Results See results from "Phenol/Chloroform purification of RNA from in-vitro transcription" from 11.05.17 for gel picture. Upconcentration fo Cas13a Lbu and Cas13 Lsh after dialysis Protocol: Äkta protein purification Participants: Ludwig, Chris Results Thursday 11th Test of different lysis methods for Phenol/Chloroform total RNA extraction. Protocol: Phenol/Chloroform purification of RNA Participants: Patrick, Julian Observations: Three lysis method were used: sonification, lysozyme digestion and heat lysis in SDS. Lysozyme digestion: according to the total RNA purification with Norgen Kit protocol. Heat lysis: Sample was incubated in 2% SDS at 75 °C for 15 min. The samples were incubated at -80 °C overnight and the purification finished the next day. Results See results from "Phenol/Chloroform purification of RNA from in-vitro transcription" from 11.05.17. Phenol/Chloroform purification of RNA from in-vitro transcription Protocol: Phenol/Chloroform purification of RNA Participants: Dawafuti, Sven Observations The samples were incubated at -80 °C overnight and the purification finished the next day. Results Friday 12th Urea PAGE of in vitro transcription samples Protocol: UREA PAGE Participants: Sven Results Monday 15th Preparation fo buffers for Heparin purification of Cas13a Lbu and Cas13a Lsh Protocol: Äkta protein purification Participants: Ludwig, Chris Tuesday 16th Heparin purification of Cas13a Lbu and Cas13 Lsh Protocol: Äkta protein purification Participants: Ludwig, Dawa Results Tuesday 23rd PCR of Cas13a Lwa part1 and part2 and backbone pSB1C3_ccdB Protocol: PCR Participants: Ludwig, Chris, Rob Observations: Q5 polymerase was used Primers Lwa part1 and part2: VF and VR Primers pSB1C3_ccdB: p-pSB-GG-Fwd, p-pSB-GG-Rev Results Assembly of Cas13a Lwa part1 and part2 with pSB1C3_ccdB using GoldenGate Cloning Protocol: GoldenGate Assembly Participants: Ludwig, Chris, Rob Observations: Insert to vector ratio: 2:1 and 3:1 Electro Transformation of assembled Lwa parts and pSB1C3 into DH5α and plating on LBCm agar plates Protocol: Electro Transformation Participants: Ludwig, Chris, Rob Observations: Next day: Only a few colonies. Electro Transformation of TEV biobricks (pSB1C3-BBa_K1319008 and pSB1C3-BBa_K1319004) and plating on LBCm agar plates Protocol: Electro Transformation Participants: Ludwig, Chris, Rob Wednesday 24th Repeat: Assembly of Cas13a Lwa part1 and part2 with pSB1C3_ccdB using GoldenGate Cloning Protocol: GoldenGate Assembly Participants: Ludwig, Erika, Dawa, Chris Observations: Insert to vector ratio: 2:1 and 3:1 Electro Transformation of assembled Lwa parts and pSB1C3 into DH5α and plating on LBCm agar plates Protocol: Electro Transformation Participants: Ludwig, Erika, Dawa, Chris Repeat: PCR of Cas13a Lwa part1 and part2 and backbone pSB1C3_ccdB Protocol: PCR Participants: Ludwig, Erika, Dawa, Chris Observations: Q5 polymerase was used Primers Lwa part1 and part2: VF and VR Primers pSB1C3_ccdB: p-pSB-GG-Fwd, p-pSB-GG-Rev Results Preparation of 5 ml LBCm overnight culture of one picked DH5α pSB1C3-Cas13a-LwaLwa colony (from first GoldenGate assembly) Protocol: - Participants: Ludwig, Erika, Dawa, Chris Thursday 25th Miniprep of DH5α pSB1C3-Cas13a-Lwa overnight culture Protocol: Minipreparation Participants: Dawa, Chris Observations: pSB1C3-Cas13a-Lwa was then used for an analytical restriction digest and was sent for sequencing (with primer VR) Analytical restriction digest with pSB1C3-Cas13a-LwaLwa with XbaI (single digest) and XbaI + SpeI (double digest) Protocol: Restriction digest Participants: Dawa, Chris Results Repeat: PCR of Lwa part2 and also GoldenGate was redone with new amplified Lwa part2 Protocol: PCR and GoldenGate Participants: Observations: PCR Primers: VF, VR Insert:Vecor 3:1 (GoldenGate) Results Friday 26th Transformation of 1 µl GoldenGate sample in DH5α and plating on LBCm agar plates Protocol: Transformation Participants: Ludwig, Dawa, Chris Observations: No colonies visible PCR of BBa_K1319004 and BBa_K1319008 (TEV) to check if biobricks are in plasmid Protocol: PCR Participants: Ludwig, Dawa, Chris Results Electro transformation of BBa_K1319004 and BBa_K1319008 (TEV) in DH5α and plating on LBCm agar plates Protocol: Participants: Observations: No colonies for BBa_K1319008 Monday 29th Chemical transformation of BBa_K1319008 (TEV) in DH5α and plating on LBCm agar plates Protocol: Chemical Transformation Participants: Ludwig, Dawa, Erika Observations: 3 µl (= 750 pg) were used Chemical transformation of GolenGate sample from 24.05. and also from 26.05. in DH5α and plating on LBCm agar plates Protocol: Chemical Transformation Participants: Ludwig, Dawa, Erika Observations: 5 µl for each transformation were used Tuesday 30th Colony PCR of DH5α pSB1C3-Cas13a-LwaLwa (from GoldenGate) Protocol: cPCR Participants: Chris, Dawa Results Colony PCR of DH5α pSB1C3-BBa_K1319004 and pSB1C3-BBa_K1319008 (TEV) Protocol: cPCR Participants: Chris, Dawa Observations: Primers: VF, VR Overnight cultures from cultures that showed right cPCR bands were set up Results Wednesday 31st Preparation of buffers for Cas13a purification (Elution, Wash, Ion exchange and SEC) Protocol: Äkta purification Participants: Milica, Ludwig, Flo, Sven, Jorge Observations: Minipreparation of DH5α pSB1C3-BBa_K1319004 and pSB1C3-BBa_K1319008 (TEV) overnight cultures Protocol: Miniprep Participants: Milica, Ludwig, Flo, Sven, Jorge Observations: Each construct was sent for sequencing with primers VR and VF Thursday 1st Colony PCR of DH5α pSB1C3-Cas13a-Lwa Protocol: cPCR Participants: Ludwig, Erika, Sven Observations: Primers: VR, VF Results Äkta purification of Cas13a Lbu and Cas13a Lwa (Affinity chromatography) Protocol: Äkta protein purification Participants: Ludwig, Erika, Sven Results Friday 2nd Total RNA extraction of E. coli W3110 with Norgen kit Protocol: Total RNA Purification with Norgen Kit Participants: Jorge Observations: Instead of using TE and lysozyme to lyse the cells, they were resuspenden in 2% SDS in TAE and incubated at 75 ºC for 15 min. The 6 samples were stored at -80 ºC and labeled T RNA #1-6 Monday 5th Preparation of overnight cultures from Rosetta pSB1C3-His-MBP-Lbu-Cas13a-WT and Rosetta p2CT-His-MBP-Lsh-Cas13a-WT glycerol stocks, DH5α pSB-Cas13a-Lwa cPCR colony 5 and DH5α pSB1C3-BBa_K1319004 cPCR colony 1 and DH5α pSB1C3-BBa_K1319008 cPCR colony 1 Protocol: - Participants: Sven RNA Adsorption Test on first 3D print Participants: Jorge Notes: Total RNA sample 5 used, prepared on Friday 02/06 by Jorge (Concentration: 126 ng/µl) Procedure: Take 1 µl presample from stock ; add with 2 µl nf H2O and 3 µl 2x RNA LD Put 5 µl of sample on device Take 1 µl of sample immediately, add with 2 µl nf H2O and 3 µl 2x RNA LD Wait 15 minutes Take 1 µl of sample immediately, add with 2 µl nf H2O and 3 µl 2x RNA LD Store at -80 °C What still needs to be done: Cook samples at 95 °C for 10 minutes and put directly on ice afterwards (to prevent refolding) Load on Gel and quantify Tuesday 6th Inoculation of Rosetta pSB1C3-His-MBP-Lbu-Cas13a-WT and Rosetta p2CT-His-MBP-Lsh-Cas13a-WT overnight cultures in each 1 l LBCarb + Cm medium Protocol: Participants: Sven, Patrick Preparation of glycerol stocks for DH5α pSB1C3-BBa_K1319004 cPCR and DH5α pSB1C3-BBa_K1319008 Protocol: Glycerol cryo stocks Participants: Sven, Patrick Minipreparation of DH5α pSB-Cas13a-Lwa cPCR colony 5, DH5α pSB1C3-BBa_K1319004 and DH5α pSB1C3-BBa_K1319008 Protocol: Miniprep Participants: Sven, Patrick Wednesday 7th Harvesting of bacteria with overexpressed Cas13a Lbu and Cas13a Lsh Protocol: Äkta purification Participants: Ludwig Preparation of 5 ml LBCm overnight culture of DH5α pSB-Cas13a-Lwa cPCR colony 5 Protocol: - Participants: Jorge Thursday 8th Preparation of wash and elution buffer for Cas13a protein purification Protocol: Äkta protein purification Participants: Ludwig, Erika Observations: pH was checked and adapted after adding of TCEP Purification of Cas13a Lbu and Cas13a Lsh (Affinity chromatography) Protocol: Äkta protein purification Participants: Ludwig, Erika, Sven Results Minipreparation of DH5α pSB-Cas13a-Lwa cPCR colony 5 and preparation of glycerol stock Protocol: Minipreparation and glycerol stock Participants: Ludwig, Erika, Sven Friday 9th PCR of pSB1C3-BBa_K1319008 (TEV) for adding a His tag Protocol: PCR Participants: Chris Observations: Primers: p-TEV-His-fwd and p-TEV-His-rev Procedure was follwed by PCR cleanup and DpnI digestion Results Monday 12th Purification of Cas13a Lbu and Cas13a Lsh (Size exclusion chromatography) Protocol: Äkta purification Participants: Sven, Ludwig, Benedikt, Milica Observations: Cas13a Lbu and Cas13a were upconcentrated to 1 ml each (centrifugal filters: MWCO: 30 kDa). 2 runs for each protein were necessary Results PCR of Lwa part2b (reordered gBlock without internal BsaI restriction site) Protocol: Participants: Sven, Ludwig, Benedikt, Milica Observations: Primers: VF, VR Procedure was follwed by PCR cleanup Results Assembly of Lwa part1, part2b and pSB1C3-ccdB with GoldenGate and transformation in DH5α Protocol: GoldenGate Cloning Participants: Sven, Ludwig, Benedikt, Milica Ligation of pSB1C3-BBa_K1319008 (TEV) after adding the His tag by overhang PCR Protocol: Participants: Sven, Ludwig, Benedikt, Milica Observations: Procedure was follwed by chemicial transformation in DH5α and plating on LbCm agar plates Results Tuesday 13th Colony PCR of DH5α pSB-Cas13a-Lwa from GoldenGate assembly (with part2b) Protocol: Participants: Ludwig, Chris Results cPCR showed positive results. Cloning seemed to work, although there was the internal BsaI restriction site Redone: Transformation of 15 µl ligation sample (of pSB1C3-BBa_K1319008 (TEV) after adding the His tag by overhang PCR) Protocol: Participants: Ludwig, Chris Observations: Colonies were visible the next day this time. 2 colonies were picked and resuspended in each 5 ml LBCm for overnight cultures Preparation of 5 ml LBCm overnight culture of DH5α pSB-Cas13a-Lwa cPCR colony 5 Protocol: Participants: Observations: Inoculation from cyro stock Results Lysis-tests with acidic phenol chlorophorm extraction Protocol: Alkaline lysis Protocol: Phenol-Chlorophorm extraction Protocol: Trizol Reagenz protocol Participants: Julian, Patrick Notes: Three lysis methods were used: Alkaline Lysis, heat lysis with SDS and Trizol-Lyse. No vortexing for lysis (may break up cells and won't be available in our device), only room-temperature (apart form SDS as reference) Cells had OD of 5.97 -> 4.78*109 cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158 Cells had OD of 5.97 -> 4.78*109 cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158 -> dilluted 1:100 (to V=10mL) and pelleted 209 miL to get to 107 cells A B C D 1 Method SDS NaOH TRIzol 2 prepare 1 mL TES (10mM Tris-HCl, 10mM EDTA, pH 8.0, 2% SDS -> TE-Buffer + SDS... 3 ----Lysis 1x 3x 1x 4 separate to tubes Spin 300xG, 5 min ->Ice, TROzol to -20! (100 miL at OD 1 -> 107 cells, Max. according to TRIzolPaper 100 miL bactSusp 100 miL bact-susp 100 miL bact-suspension 5 No washing step, bad for mRNA (TRIzolP.) 300 µl (0.45 ml) TES resuspend pellet 6 12,5 uL Inhibitor (40k U/ml, 1 U/miL required) 15 min, 75 °C lysis, vortex one probe after another 7 (Addition of inhibitor); Transform total volume to gel tube resuspend thorougly in 66 µl (100 miL) Solution1 8 132 µl (200 miL) Sol2, INVERT 4x 9 wait 0, 1 ,3 min 10 add 99 µl (150 miL) Sol3, INVERT 4x 11 Total volume: 297 µl (0.45 ml) 12 --phenChloExtr 13 measure pH, add acid... meassure Trizol PH, add acid if needed (pH <5) 14 add 600 µl [same V (0.45 ml)] Phe:chloro. add 1 mL TRIzol to 107 cells 15 5 min incubation 16 add 0.2 mL CHLOROPHORM 17 incub 2.5 (2-3) min, RT 18 centri 12k g 15 min 4°C 19 angle 45°, extract top phase carefully -> discard everything else, prot & DNA -> which Vol for the TRIzol aqueous phase? 20 add 0.4 (0.375) ml Isopropanol, wait 10 min, RT 21 centrifuge 10 min 12k , 4°C -> 22 Aditional step, because RNA pellet was not entierly spinned down: Centrifugation (16000 rcf, 5 min, 4 °C) big-pellet assay in fridge, but 1. interphase was punctured/gel(DNA?) stuck to pipette 2. s.u. 23 Aditional step, because suspected RNA fog was not entirely spinned down: remove upper and lower RNA fog phase 24 Additional step: Centrifugation (16000 rcf, 5 min, 4 °C) 25 No RNA pellet or RNA fog any more :( 26 Addition of 400 µl isopropanol -> No RNA pellet or fog any more ->prob. removed too much 27 discard supernatant -> RNA in gel-like pellet! -> gel hardly visible, nearly draged it out of tube (stuck to pipette...) 28 resuspend in 0.75 ml 70 % (75% ideally) ethanol -> storeed RNA at -20°C is stable up to 1 year... 29 vortex, centri 5 min 7,5k 4° 30 discard supernatant, air dry for 5-10 min (do NOT let completely dry out) 31 resuspend in RNAse free water 30 µl (20-50 miL) ->measurement... Results No pellets seen during procedure, concentration too low for photometer -> repeat with more cells Wednesday 14th Up-concentration of Cas13a Lbu and Cas13 Lsh after SEC and concentration measurement Protocol: Participants: Chris, Patrick Observations: Centrifual filters: MWCO: 30 kDa Concentrations were measured using a Bradford assay Results Cas13 Lbu: 746 mg/ml, Cas13 Lsh: 120 mg/ml Miniprep of overnight culture of DH5α pSB-Cas13a-Lwa cPCR colony 5 Protocol: Minipreparation Participants: Chris, Patrick Observations: Plasmid was again sent for sequencing with VR and VF Results First sequencing looked good Friday 16th Sending pSB-Cas13a-Lwa cPCR colony 5 for sequencing again with more primers Protocol: Sequencing Participants: Chris, Jorge Observations: Primer: p-seq-lwa 1-4 Preparation of first readout experiment with Cas13 Lbu and Cas13 Lsh Protocol: Readout Participants: Chris, Jorge Observations: Cleavage activity was tested with Aptamers (Malachite, Spinach) and RNase alert Results The Cas13a proteins were activated and cleaved the aptamers. This resulted in a decrease in fluorescence intensity. Lysis-tests with acidic phenol chlorophorm extraction Protocol: Lysis test pipetting scheme Protocol: Phenol-Chlorophorm extraction Participants: Julian, Patrick Notes: Cell suspension had OD600 (extrapolated) of 2,220 -> 1.78 x 109 cells/ml according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158 Experiments were done with 108 and 109 cells per sample, because it failed with 107 cells last time Aspiration of supernatant in phenol-chlorophorm extraction: about 2/3 aspirated (200 µl) and 1/3 (100 µl) left to not destroy interphase No pellet after RNA precipitation (-80 °C) and subsequent centrifugation in: NaOH, 1 min, 108 SDS, 109 Trizol, 108 Mistakes: SDS, 109: aspirated 100 µl instead of 200 µl supernatant NaOH, 108, 3 min: contamination with DNA (<10% DNA interphase transfered) NaOH, 109, 3 min: added + 200 µl of SDS 109 tube ! Did not calibrate alkaline lysis solution 3 to given pH -> probably more RNA degradation ! Did store RNA at -20 °C instead of -80 -> probably more RNA degradation Results Sample [] [µg/ml] adjusted C** A260/A280 A260/A230 A230 [A] A260 [A] A280 [A] A320 [A] 1 SDS, 108 cells 36.8 55 1.704 2.044 0.047 0.094 0.056 0.002 2 SDS, 109 cells* 119 357 1.693 2.069 0.146 0.300 0.178 0.002 3 NaOH, 0 min, 108 cells 35.2 53 1.725 2.146 0.042 0.089 0.052 0.001 4 NaOH, 0 min, 109 cells 586 879 1.923 1.993 0.740 1.470 0.767 0.005 5 NaOH, 1 min, 10^8 cells 73.6 110 1.235 0.586 0.317 0.187 0.152 0.003 6 NaOH, 1 min, 109 cells 370 555 1.945 1.958 0.475 0.927 0.478 0.003 7 NaOH, 3 min, 108 cells* 259 389 1.849 1.491 0.439 0.652 0.355 0.005 8 NaOH, 3 min, 109 cells* 275 413 1.850 2.212 0.316 0.693 0.368 0.005 9 TRIzol, 108 cells 194 265 1.362 0.221 2.200 0.487 0.358 0.002 10 TRIzol, 109 cells 160 218 1.429 0.488 0.822 0.403 0.283 0.003 Measured [RNA] by Implen Nanophotometer (20170616). **adjusted for different supernatant V aspirated at extraction step see also "Denaturing PAGE for ssRNA" Monday, the 26th Monday 19th Preparation of 5 ml LBCm overnight culture of DH5α pSB-Cas13a-Lwa cPCR colony 5 Protocol: Overnight culture Participants: Ludwig Observations: Inoculation from cryo stock Preparation of 5 ml LBCm overnight culture of DH5α pSB1C3-BBa_K1319008-His (TEV) 1 and 2 Protocol: Overnight culture Participants: Ludwig Observations: Inoculation from cryo stock Electro transformation of pSB1C3-Cas13a-Lwa (cPCR colony 5) into E. coli BL21star Protocol: Electro transformation Participants: Ludwig, Chris Observations: pSB1C3-Cas13a-Lwa (cPCR colony 5) was verified by sequencing and is named now pSB1C3-Cas13a-Lwa Tuesday 20th Minipreparation of DH5α pSB-Cas13a-Lwa cPCR colony 5 Protocol: Minipreparation Participants: Ludwig, Chris Observations: The plasmid was sent for sequencing with VR, VF, p-seq-lwa 1-4 Minipreparation of DH5α pSB1C3-BBa_K1319008-His (TEV) 1 and 2 Protocol: Minipreparation Participants: Ludwig, Chris Observations: The plasmids were sent for sequencing with VR, VF Preparation of 5 ml LBCm overnight culture of one picked BL21star pSB1C3-Cas13a-Lwa colony Protocol: Overnight culture Participants: Ludwig, Chris Wednesday 21st Minipreparation of BL21star pSB1C3-Cas13a-Lwa overnight culture and send for sequencing Protocol: Minipreparation Participants: Erika, Igor, Milica Observations: Primers: VF, VR, p-seq-lwa 1-4 Preparation of glycerol stock for BL21star pSB1C3-Cas13a-Lwa Protocol: Glycerol stock Participants: Erika, Igor, Milica Friday 23rd Time-series of alkaline lysis, in triplets Protocol: Lysis test pipetting scheme Participants: Julian, Patrick Notes: Did alkaline Lysis with inbubation/lysis times of 0 min (~10 sec ), 1 min, 3 min, 10 min, Heat+SDS as reference, no Trizol Cell suspension with OD600 (extrapolated) of 2.182 -> 1.78 x 109 cells/ml Phenol-Chlorophorm Precipitation at -80 was done until Wednesday Results see "Denaturing PAGE for ssRNA" Thursday (not Monday!), the 29th Monday 26th Gel analysis of extracted RNA (July 16th !) Protocol: Denaturing PAGE for ssRNA Participants: Julian, Patrick Notes: see Friday, 16th Results Tuesday 27th Electro Transformation of pSB1C3-BBa_K1319008-His (TEV) in BL21star and plating on LBCm agar plates Protocol: Electro transformation Participants: Ludwig, Chris Observations: 1 µl was used Preparation of 5 ml LBCm + Carb overnight culture of E. coli Rosetta p2CT-His-MBP-Lbu-Cas13a-WT Protocol: Overnight culture Participants: Ludwig, Chris Preparation of 5 ml LBCm overnight culture of E. coli DH5α pSB1C3-BBa_K1319008-His 2 Protocol: Overnight culture Participants: Ludwig, Chris Wednesday 28th Inoculation of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT in 4x 1 l 2xYTCm + Carb medium for expression of Cas13a Lbu Protocol: Äkta protein purification Participants: Chris, Ludwig,Jorge Minipreparation of DH5α pSB1C3-BBa_K1319008-His 2 and send for sequencing Protocol: Minipreparation and Sequencing Participants: Chris, Ludwig, Jorge Observations: Primers: VF, VR Preparation of 5 ml LBCm overnight culture of BL21star pSB1C3-BBa_K1319008-His from picked colony Protocol: Overnight culture Participants: Chris, Ludwig, Jorge Plating of DH5α pSB1C3-Cas13a-Lwa glycerol stock on LBCm agar plates Protocol: Participants: Observations: Colonies should be checked if they grow the same and a cPCR should confirm that there is only one plasmid Thursday 29th Preparation of glycerol stock of BL21star pSB1C3-BBa_K1319008-His Protocol: Glycerol stock Participants: Ludwig, Chris Observations: From this glycerol stock a 5 ml LBCm overnight culture was prepared A 5 ml LBCm overnight culture was prepared directly from this cryo stock Harvesting of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT with overexpressed Cas13a Lbu Protocol: Äkta protein purification Participants: Ludwig, Chris Observations: The pellet was stored at -80 °C Colony PCR of DH5α pSB1C3-Cas13a-Lwa (plated glycerol stock) Protocol: cPCR Participants: Ludwig, Chris Results Time-series of alkaline lysis, in triplets (continued) Protocol: Phenol-Chlorophorm extraction Participants: Patrick Notes: Continuing after -80 °C precipitation Extremely big pellets after 99 % EtOH precipitation for all samples with SDS: Possibly contaminated with DNA Results SDS 10⁹ seems to have lower yield than NaOH -> maybe more SDS? (10⁸ has no problems...) NaOH is done after "0" minutes (about 10 sec for adding base to all eppis and turning 3 times) Sample Number of sample [] [µg/ml] A260/A280 A260/A230 A230 [A] A260 [A] A280 [A] A320 [A] 1 SDS, 108 cells 1 62.4 2.080 0.118 1.343 0.174 0.093 0.018 2 2 56.8 2.029 0.128 1.123 0.153 0.081 0.011 3 3 47.6 1.983 0.102 1.178 0.127 0.068 0.008 4 SDS, 109 cells 1 354 1.782 >0.4 >2.5 0.913 0.525 0.029 5 2 260 1.857 0.295 2.222 0.665 0.365 0.015 6 3 376 1.801 0.392 2.422 0.962 0.544 0.022 7 NaOH, 0 min, 108 cells 1 39.2 1.690 2.042 0.056 0.106 0.066 0.008 8 2 53.6 1.614 1.887 0.086 0.149 0.098 0.015 9 3 68.8 1.720 2.457 0.070 0.172 0.100 0.000 10 NaOH, 0 min, 109 cells 1 629 1.911 2.466 0.643 1.578 0.828 0.005 11 2 17.6 1.692 2.200 0.019 0.043 0.025 -0.001 12 3 619 1.897 2.453 0.636 1.553 0.821 0.005 13 NaOH, 1 min, 108 cells 1 52 1.806 2.203 0.059 0.130 0.072 0.000 14 2 33.2 1.729 2.371 0.035 0.083 0.048 0.000 15 3 58.4 1.738 2.393 0.061 0.146 0.084 0.000 16 NaOH, 1 min, 109 cells 1 606 1.893 2.469 0.620 1.522 0.807 0.006 17 2 627 1.898 2.431 0.651 1.574 0.832 0.006 18 3 22.4 1.806 2.435 0.023 0.056 0.031 0.000 19 NaOH, 3 min, 108 cells 1 44 1.864 2.444 0.044 0.109 0.058 -0.001 20 2 61.2 1.779 2.593 0.058 0.152 0.085 -0.001 21 3 5.6 1.400 2.800 0.004 0.013 0.009 -0.001 22 NaOH, 3 min, 109 cells 1 619 1.908 2.432 0.643 1.554 0.818 0.007 23 2 570 1.927 2.468 0.583 1.430 0.745 0.006 24 3 20.4 1.759 2.429 0.020 0.050 0.028 -0.001 25 NaOH, 10 min, 108 cells 1 10.8 1.588 2.250 0.012 0.027 0.017 0.000 26 2 8.8 1.571 3.143 0.006 0.021 0.013 -0.001 27 3 4.4 1.375 3.667 0.002 0.010 0.007 -0.001 28 NaOH, 10 min, 109 cells 1 503 1.916 2.499 0.508 1.262 0.661 0.005 29 2 564 1.911 2.487 0.572 1.415 0.743 0.005 30 3 611 1.902 2.467 0.625 1.533 0.809 0.006 Friday 30th Minipreparation of BL21star pSB1C3-BBa_K1319008-His Protocol: Minipreparation Participants: Max Observations: The plasmid was sent for sequencing with primers VR and VF Gel analysis of alkaline-extracted RNA time-series (July 23th !) Protocol: Denaturing PAGE for ssRNA Participants: Julian, Patrick Notes: see Friday, 23th Results Monday 3rd Preparation of 5 ml LBCm overnight culture of BL21star pSB-Cas13a-Lwa Protocol: Overnight culture Participants: Max Tuesday 4th Inoculation of BL21star pSB-Cas13a-Lwa in 4x 1 l 2xYTCm medium for expression of Cas13a Lwa Protocol: Ni NTA agarose protein purification Participants: Max, Dawa Purification of Cas13a Lbu Protocol: Ni NTA agarose purification Participants: Max, Dawa Observations: Fresh DTT was added to the lysis buffer 1 ml Ni-NTA agarose was used per 4 ml lysate Sample was incubated 1 h at 4 °C while shaking The procedure was followed by TEV cleavage overnigth while dialyzing Results Wednesday 5th Harvesting of BL21star pSB-Cas13a-Lwa with overexpressed Cas13a Lwa Protocol: Ni NTA protein purification Participants: Max, Dawa, Erika Observations: Pellet was incubated for 1 h at -80 °C before it was used for purification Results Purification of Cas13a Lwa Protocol: Ni NTA agarose purification Participants: Max, Dawa, Erika Observations: Fresh DTT was added to the lysis buffer 1 ml Ni-NTA agarose was used per 4 ml lysate Sample was incubated 1 h at 4 °C while shaking Results Precipitation of TEV plasmid for European meetup in Delft Protocol: - Participants: Max, Dawa, Erika Observations: pSB1C3-BBa_K1319008-His was lyophylized by Ethanol precipitation and evaporation Friday 7th Reverse Ni-NTA purification of cut Cas13a Lbu Protocol: Ni NTA agarose protein purification Participants: Rob Observations: Ni NTA agarose binds the cleaved His-MBP tag, Cas13a Lbu is in the flow-through Results Monday 10th Preparation of 50 ml LBCm overnight culture of BL21star pSB1C3-BBa_K1319008-His (TEV) Protocol: Overnight culture Participants: Rob, Ludwig, Rob Tuesday 11th Inoculation of BL21star pSB1C3-BBa_K1319008-His (TEV) in 3x 1 l 2xYTCm medium for expression of the TEV protease Protocol: Äkta protein purification Participants: Ludwig, Max Observations: Induction with 1 mM IPTG at OD600 of 0.6 Expression at 16 °C overnight RNase alert assay with Cas13a Lbu and Cas13a Lsh Protocol: Cas13a activity assay Participants: Igor, Rob Results Wednesday 12th Harvesting of BL21star pSB1C3-BBa_K1319008-His with overexpressed TEV protease Protocol: Äkta protein purification Participants: Ludwig, Milica Friday 14th Protein purification of TEV protease Protocol: Äkta protein purification Participants: Ludwig, Max, Sven Results Cas13a activity assay with RNase alert Protocol: Cas13 activity assay Participants: Dawa, Rob Results RNA Extraction with commercial kit Participants: Patrick Notes: used ReliaPrep miRNA Cell and Tissue Miniprep System (Promega) according to instructions Specification: Use in range of 102 to 106 cells. Tested 106 and 108 cells Results No nanodrop-measureable amount of RNA/DNA with 106 cell, <10 ug/ml RNA with 108 cells Terrible yield, repeat again to be sure Monday 17th Cas13a activity assay Protocol: Cas13a activity assay Participants: Igor Observations: Results RNA Extraction with commercial kit Participants: Patrick Notes: used ReliaPrep miRNA Cell and Tissue Miniprep System (Promega) according to instructions Specification: Use in range of 102 to 106 cells. Tested 106 and 108 cells Results Again no measureable amount of RNA for 106 cells. For 108 cells, RNA concentration was increased through column-binding and elution (small elution volume) Elution seems effective, second elution has only 20 % of first elution's amount of RNA Tuesday 18th Preparation of 40 ml LBCm + Carb overnight culture of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT Protocol: Äkta protein purification Participants: Chris, Max, Dawa Wednesday 19th Cas13a activity assay Protocol: Cas13a activity assay Participants: Igor Observations: Results Inoculation of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT in 3x 1 l 2xYTCm + Carb medium for expression of Cas13a Lbu Protocol: Äkta protein purification Participants: Max, Dawa Thursday 20th Size exclusion chromatography of affinity purified TEV protease Protocol: Äkta protein purification Participants: Max, Sven, Ludwig Results Harvesting of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT with overexpressed Cas13 Lbu Protocol: Äkta protein purification Participants: Max, Sven, Ludwig In vitro Transcription of target RNA, Lbu crRNA and Lsh crRNA Protocol: In vitro Transcription Participants: Igor Observations: Procedure was followed by DNAase treatment and phenol chloroform extraction Results Friday 21st PCR of gBlock of aeBlue Protocol: PCR Participants: Chris Monday 24th Digestion of insert and pSB1C3 with Pst1-HF and EcoRI-HF Protocol: Digestion and Ligation Participants: Chris Observations: Plasmid was dephosporylated with arctic phosphatase and insert was ligated with plasmid overnight at 16 °C Tuesday 25th Electro transformation of aeBlue ligation sample in E. coli Turbo cells Protocol: Electro transformation Participants: Max Wednesday 26th Colony PCR of plated pSB1C3-aeBlue Protocol: cPCR Participants: Max Results Preparation of 5 ml LBCm overnight cultures (picked colonies of aeBlue ligation) Protocol: Overnight culture Participants: Max Thursday 27th Minipreparation of Turbo pSB1C3-aeBlue overnight cultures and send for sequencing Protocol: Minipreparation and sequencing Participants: Max Friday 28th Upconcentration of TEV protease to 2 mg/ml and stored in TEV storage buffer Protocol: Äkta protein purification Participants: Max Monday 31th Tuesday 1st Intein-Extein-Readout Protocol: DNA Gel-Purification and Genomic DNA Purification Participants: Max Observations: Purification of b-Gal fragments from 1 % Agarose Gel 60 µl per lane Genomic DNA purification from E.coli W3110+ Results DNA was lost during the purification Wednesday 2nd Intein-Extein-Readout Protocol: Q5-PCR and PCR-Cleanup kit Participants: Max Observations: Repeated PCR of beta-Gal N and C Terminal fragments from 31.7. 3 different Templates: W3110 genomic DNA W3110 genomic DNA diluted 1:100 W3110 2 µl of cryostock Clean-up of PCR with Purification Kit and elution in 33 µl nf water InterLab Study calibration Protocol: IGEM HQ InterLab Study Protocol Participants: Dawafuti Erika Observations: Patch length correction was on in the plate reader. Calibration for the plate reader experiment done with LUDOX and Fluorescein. We saw no difference between LUDOX and water when measuring at an OD of 600 nm. For the fluorescein measurement, the best gain was 600 using 485 nm/520 nm. Plasmids from the plate 7 (positive and negative control, and devices 1-6) were resuspended. Thursday 3rd Intein-Extein-Readout Protocol: Agarose-Gel Participants: Max Observations: Gel of purified PCR 1,5% small Gel, 120 V, 30 minutes Results PCR with undiluted extracted DNA didn't work. Monday 7th Protein-Purifcation of Lbu and Lsh Protocol: - Participants: Max, Ludwig, Teeradon Observations: Inoculation of main cultures of Rosetta p2CT-His-MBP-Lbu_C13a_WT and Lsh in LB(Carb+Cm) InterLab Study Day 1: Transformation Protocol: IGEM HQ InterLab Study Protocol Participants: Dawafuti Erika Observations: Each plasmids was transformed two times: with chemical transformation and with electroporation, for a total of 16 plasmids. Results Every plate except the ones for Device 1 had colonies. Tuesday 8th First test with 3D printed 96-well plate Protocol: - Participants: Max Observations: Tested different concentrations of fluorescein in a 3D printed 96 well plate on paper. Scanned at differnt gains at a constant focal height. Dilutions were 1:20; 1:400 and 1:8000 of Fluorescein-FITC in water Results No light bleeding was observed in the experiment InterLab Study Day 2: Colony transfer to liquid culture Protocol: IGEM HQ InterLab Study Protocol Participants: Dawafuti Erika Observations: Device 1 was again transformed, as no colonies grew on the plate. Liquid cultures for the 7 plasmids whose plates showed colonies (all except Device 1) were made in duplicate. The plates were stored at -4 °C. Wednesday 9th Expression and Harvesting of Cas13a variants Protocol: Protein Purification Participants: Max, Ludwig Observations: Harvested Cas13a Lbu overexpression. Inoculated 2 litres 2xYT media with Rosetta Cas13a Lsh and let them grow at 16 °C overnight with 1 mM IPTG InterLab Study Day 3: Plate reader experiments Protocol: IGEM HQ InterLab Study Protocol Participants: Dawafuti Erika Observations: After measuring the OD600 of the O/N cultures, they were diluted with LB+Cm to an OD600 of 0.02, in duplicates. The absorbance and fluorescence of the diluted cultures were measured at 0, 2, 4 and 6 hours after the cultures were set. Patch length correction was on in the plate reader. Device 1 was again transformed, as no colonies grew on the plate. Results Transformation of Device 1 was successful. Thursday 10th His Purification of Cas13a Lbu and Harvesting of Cas13a Lsh Protocol: His-Purification Participants: Max, Ludwig, Milica, Sven, Benedikt Observations: His purification of Cas13a Lbu according to protocol. Elution in 4 times 4 ml. ON Dialysis with TEV protease in 2 litres of Lysis buffer. Harvesting of Cas13a Lsh according to protocol InterLab Study Day 4: Preparation of O/N cultures for plate reader experiments Protocol: IGEM HQ InterLab Study Protocol Participants: Dawafuti Erika Observations: O/N liquid cultures in duplicate were set up from all 8 Devices. Annealing of Invitro Transcription Targe Inhibitor Circuit 3a (IC3a) Protocol: Participants: Florian Observations: Invitro Transcription of IC3a Protocol: In vitro transcription Participants: Florian Observations: Incubated over night at 37 ºC Results See results of PEC from 11.10.2017 Friday 11th Reverse HIS purification and SEC of Cas13a Lbu Protocol: His purification and SEC protein purification Participants: Ludwig, Benedikt, Max, Sven Observations: Incubation of cleaved Lbu sample with Ni-NTA beads (ca. 7 mL) (1 h, 4 °C) Application on column, keep FT (there is our protein) Elution of His-MBP with ca. 10 mL Elution buffer (for gel analysis) Washing of Ni-NTA bead (3x with water) and storage in 20 % EtOH Up-concentration of Cas13a Lbu and buffer exchange in SEC buffer (centrifugal filter: MWCO: 30 kDa, 4 °C, 5000 RCF) Results See Gel image of 14.8. for results. o Protocol: Restriction digest, Phenol-chlorophorm purification of RNA Participants: Florian Observations: Phenol-Chloroform with the wrong pH was used. Results Sample was lost due to the use of the wrong Phenol-Chloroform solution. InterLab Study Day 5: Plate reader calibration and experiments Protocol: IGEM HQ InterLab Study Protocol Participants: Dawafuti Erika Observations: Patch length correction was off in the plate reader. Calibration for the plate reader experiment done with LUDOX and Fluorescein. We saw the difference in measurements between LUDOX and water. After measuring the OD600 of the O/N cultures, they were diluted with LB+Cm to an OD600 of 0.02, in duplicates. The absorbance and fluorescence of the diluted cultures were measured at 0, 2, 4 and 6 hours after the cultures were set. Results Monday 14th SDS PAGE of Lbu purifcations Protocol: SDS-PAGE Participants: Ludwig Observations: SDS-PAGE of Lbu purification from last week Invitro Transcription of IC3a Protocol: In vitro transcription Participants: Florian Observations: Results See results of Restriction digestion and PCE from 15.08.17 Tuesday 15th DNAse I digest and PCE of the RNA Protocol: Restriction digest, Phenol-chlorophorm purification of RNA Participants: Florian Observations: Phenol-Chloroform with the wrong pH was used. Results IC3A has a length of 33 bp = band on top. Wednesday 16th Cas13a preparation Protocol: - Participants: Benedikt, Ludwig Observations: Concentration measurment of Lbu, Lsh and Lwa with Bradford essay Results Concentration of proteins measured(µg/ml): 718 - Lbu 13.6 (pooled fractions 6, 7, 8) 387 - Lbu 12.7 90,8 - Lsh 13.6 (pooled fraction 7,8,9) 379 -Lwa 5.7 (elution 1) 1620 - Lwa 5.7 (elution 2) 818 - Lwa 5.7 (elution 3) 405 -Lwa 5.7 (elution 4) Thursday 17th DNA RNA Hybrid Annealing of IC3A with Circuit 3 Activator (C3A) – IC3a concentration screen Protocol: Participants: Florian Observations: Incubation at room temperature for 1 h of DNA and RNA with varied concentrations of IC3a (RNA). Results 4% Agarose does not seem to work for separation. No oligos at all visible after staining with SybrGreen II and SybrGold. Protein-Expression of Lbu and Lwa Protocol: Protein-Expression Participants: Benedikt, Ludwig, Milica Observations: Expression of Rosetta Cas13a Lbu (3L 2xYT Medium, 16°C ON) Expression of Rosetta Cas13a Lwa (2L 2xYT Medium, 16°C ON) Friday 18th Harvesting and Purification of Cas13a Lbu and Lsh Protocol: Protein Purification Participants: Benedikt, Milica Observations: Harvesting of both expressions Purification of Cas13a Lbu according to protocol. Elution in 4 times 3 ml elution buffer. Results Sunday 20th Cas13a activity assay Protocol: Cas13a activity assay Participants: Igor Observations: Results Monday 21st Continuation of Cas13a purification Protocol: His-Purification Participants: Benedikt, Milica, Max Observations: Continued His purification of Cas13a Lbu Overnight dialysis with TEV protesae Results As visible on the gel the elutions 1 and 2 contained the highest amount of protein and were pooled for the TEV cleavage. Cas13a activity assay Protocol: Cas13a activity assay Participants: Igor Observations: Results FINA extraction for DNA with E. coli W3110 pre-purified gDNA Protocol: FINA Extraction for DNA Participants: Jorge Observations: The gDNA was pre-purified with the Phenol/Chloroform method. The gDNA solution was diluted 1:100, 1:1000, 1:100000, 1:10e6 and 1:10e8. Each dilution was then used as a sample for the FINA extraction. From each dilution, two extractions were performed. One as in protocol (1N-5N), the other without the NaOH washing step (1-5). As negative control (KK) FINA extraction was performed as in protocol with nuclease free water as sample. Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples and purified gDNA from 21.08.17 Protocol: PCR amplification with Q5 Master Mix Participants: Jorge Observations For the membranes, no template was directly pipette. Instead, the membranes were put in the tube. p-bGal_N_N was used as forward primer p-bGal_N_C was used as reverse primer Annealing was performed at 61 ºC The amplification step was 90 s long A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder Results 1N-5N: 1:100-1:10e8 dilutions FINA extraction performed as in protocol. 1-5: 1:100-1:10e8 dilutions FINA extraction without washing step. 1K-5K: 1:100-1:10e8 dilutions. KK: negative control for FINA extraction (nuclease free water as sample). Tuesday 22nd Reverse His purification of Cas13a Lbu Protocol: His purification Participants: Max, Benedikt, Milica Observations: Incubation (shaker!!) of cleaved Lbu sample with Ni-NTA beads (ca. 7 mL) (1 h, ice) Application on column, keep FT (there is our protein) (on ice) Washing with 10 mL lysis-buffer Elution of His-MBP with ca. 10 mL Elution buffer (for gel analysis) Washing of Ni-NTA bead (3x5mL with water) and storage in 20 % EtOH Results Cas13a activity assay Protocol: Cas13a activity assay Participants: Igor Observations: Results FINA extraction for DNA with E. coli W3110 cell culture Protocol: FINA Extraction for DNA Participants: Jorge Observations: The extraction was done when the culture had an OD600 of 1.2. The cells were diluted 1:1, 1:100 and 1:1000 before the extraction. Each dilution was used as sample twice: as in protocol (1N-3N and P1N-P3N) and washed with 300 ul lysis buffer from RNA extraction kit (Norgen, P/N 17200) (1G-3G and P1G-P3G). Membranes 1N-3N and 1G-3G were put in 40 ul nuclease free water and incubated at RT for 10 min in order for the bounded DNA to go into solution. Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 22.08.17 Protocol: PCR amplification with Q5 Master Mix Participants: Jorge Observations The membranes from the FINA extraction were directly put into the PCR-mix. No template DNA was directly pipetted. p-bGal_N_N was used as forward primer. p-bGal_N_C was used as reverse primer. Annealing was performed at 61 ºC. The amplification step was 90 s long. A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products. Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder Results 1N-3N: 1:1-1:1000 dilutions FINA extraction performed as in protocol, loaded in gel after elution (no PCR). 1G-3G: 1:1-1:1000 dilutions FINA extraction performed with lysis buffer as washing step, loaded in gel after elution (no PCR). P1N-P3N: 1:1-1:1000 dilutions FINA extraction performed as in protocol. P1G-P3G: 1:1-1:1000 dilutions FINA extraction with lysis buffer as washing buffer. KK: negative control for FINA extraction (nuclease free water as sample). Wednesday 23rd Cas13a purification Protocol: Size Exclusion Chromatography Participants: Benedikt, Milica Observations: Measuring concentration after Reverse His Lbu Upconcentration and buffer exchange of Lbu in 30 kDa Centricon SEC Run of Lbu according to protcol Results See SDS PAGE of 24.8. to see the SEC result. FINA extraction for DNA with E. coli W3110 cell culture Protocol: FINA Extraction for DNA Participants: Jorge, Benedikt Observations: The extraction was done when the culture had an OD600 of 2.33. The cells were diluted 1:1, 1:100, 1:10000, 1:10e6 and 1:10e8 before the extraction. Each dilution was used as a sample for the FINA extraction. In addition, three 1:1 dilutions were used as samples for FINA extraction: two according to protocol and the third with 300 ul lysis buffer as washing buffer. One of the 1:1 membranes was put in nuclease free water and incubated at RT for 20 min in order for the bounded DNA to go into solution. Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 23.08.17 Protocol: PCR amplification with Q5 Master Mix Participants: Jorge, Benedikt Observations The membranes from the FINA extraction were directly put into the PCR-mix. No template DNA was directly pipetted. p-bGal_N_N was used as forward primer. p-bGal_N_C was used as reverse primer. Annealing was performed at 61 ºC. The amplification step was 90 s long. A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products. Afte the PCR, only 2,5,L and KK were loaded in the gel, as the other tubes had no liquid. The gel was loaded before it was submerged in TAE-buffer. Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder Results 2: 1:100 dilution FINA extraction performed as in protocol L: 1:1 dilution FINA extraction performed with 300 ul lysis buffer as washing buffer. KK: negative control for FINA extraction (LB-medium as sample). Thursday 24th SDS-Gel of SEC-Run from 23.8. Protocol: SDS-PAGE Participants: Chris Observations: 10% SDS PAGE of SEC Run samples Marker; Sample prior to run; Fractions 5-16; FT Centricon Results The samples with the highest protein concentration were pooled and used for further experiments. DNA-RNA-Hybrid-Building Protocol: Participants: Florian Observations: Native PAGE for short DNA oligos Protocol: Participants: Florian Observations: Results Slight band shift of the oligo between 4 and 5 µM. Slight band shift of the oligo between 4 and 5 µM. Looks better than 30% PAGE in its resolution of the DNA oligos. Cas13a activity assay Protocol: Cas13a activity assay Participants: Igor Observations: Results FINA extraction for DNA with E. coli W3110 cell culture (Repetition of the experiment from 23.08.17) Protocol: FINA extraction for DNA Participants: Jorge, Benedikt Observations: As there were problems with the gel from the 23th, we repeated the experiment. The extraction was done when the culture had an OD600 of 2.78. The cells were diluted 1:1, 1:100, 1:10000, 1:10e6 and 1:10e8 before the extraction. Each dilution was used as a sample for the FINA extraction. In addition, three 1:1 dilutions were used as samples for FINA extraction: two according to protocol and the third with 300 ul lysis buffer as washing buffer. One of the 1:1 membranes was put in nuclease free water and incubated at RT for 20 min in order for the bounded DNA to go into solution. Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 24.08.17 Protocol: PCR amplification with Q5 Master Mix Participants: Jorge, Benedikt Observations The membranes from the FINA extraction were directly put into the PCR-mix. No template DNA was directly pipetted. p-bGal_N_N was used as forward primer. p-bGal_N_C was used as reverse primer. Annealing was performed at 61 ºC. The amplification step was 90 s long. A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products. Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder Results 1-5: 1:1-1:10e8 dilutions FINA extraction performed as in protocol L: 1:1 dilution FINA extraction performed with 300 ul lysis buffer as washing buffer. KK: negative control for FINA extraction (LB-medium as sample). Total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with Norgen kit Protocol: Total RNA Purification with Norgen Kit Participants: Jorge, Benedikt Observations: 2 tubes stored at -80 ºC. Labelled gRNA #1, gRNA #2. Results Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT Protocol: Phenol/Chloroform purification of RNA Participants: Jorge Observations: The incubation step at -80 ºC was done, O/N. 2 tubes stored at -80 ºC. Labelled gRNA PEC #1, gRNA PEC #2. Results Friday 25th Continuation of Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT from yesterday Protocol: Phenol/Chloroform purification for RNA Participants: Jorge Observations: See entry from 24.08.17 Monday 28th DNA RNA Hybrid Annealing of IC3A with Circuit 3 Activator (C3A) – IC3A concentration screen (high concentration) Protocol: Participants: Florian Observations: Results Native PAGE 20 % Cas13a activity assay Protocol: Cas13a activity assay Participants: Igor Observations: Results Tuesday 29th Intein-Extein-Readout Protocol: PCR with Q5 Polymerase, PCR Cleanup Kit, Restriction digest and Ligation Participants: Max Observations: Resuspension of gblocks Split_Uni C and Split_Uni N at a conc. of 100 nMol PCR of SPlit Uni C und Split Uni N with Q5 Polymerase Clean-up with Monarch Kit Digestion of PCR products and psB1C3 with PstI-HF und EcoRI-HF in Cutsmart buffer Dephosphorilation with Antarctic Phosphatase Ligation ON with T4 DNA Ligase FINA extraction for DNA with E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT Protocol: FINA extraction for DNA Participants: Jorge Observations: The extraction was done when the culture had an OD600 of 0.88. Na: Extraction as in protocol. Cl: Washed with 10 mM HCl instead of NaOH. H: incubated 10 min at 80 ºC. L: 10 min incubated in 1 mg/ml lysozyme. LH: 10 min incubated in 1 mg/ml lysozyme at 80 ºC. Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 24.08.17 Protocol: PCR amplification with Q5 Master Mix Participants: Jorge Observations The membranes from the FINA extraction were directly put into the PCR-mix. No template DNA was directly pipetted. p-bGal_N_N was used as forward primer. p-bGal_N_C was used as reverse primer. Annealing was performed at 61 ºC. The amplification step was 90 s long. A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products. Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder Results See Q5-PCR from 30.08.17 FINA extraction for RNA with purified total RNA (gRNA PEC #1) Protocol: FINA extraction for RNA Participants: Jorge Observations: First strand cDNA synthesis from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT purified total RNA (Phenol/Chloroform extraction from 24.08.17) and FINA-RNA eluate Protocol: First strand cDNA synthesis Participants: Jorge Observations: p-bGal_N_C was used as primer. Sample gRNA PEC #1 from 24.08.17 was diluted 1:4 and used as sample P. The eluate from the FINA extraction for RNA from 29.08.17 was used as sample S. For both reactions (S and P), 6 ul sample were pipetted. Wednesday 30th DNA and RNA hybrid of 3C-A and IC3-A Protocol: Participants: Florian Observations: Concentration screen IC3a between 1 and 5 µM. Incubation for dimerization again for 1 hour and at room temperature. Results 20% Native PAGE, 12.5 mM MgCl2 Shift of DNA-oligo band with higher concentration of IC3a RNA until 3.5 µM. No band of single DNA oligo C3a at 5 µM visible anymore. Hypothesis of full dimerization, but with a big background of single stranded IC3a RNA. Q5-PCR for beta-galactosidase from E. coli with cDNA samples from 29.08.17 Protocol: PCR amplification with Q5 Master Mix Participants: Jorge Observations: p-bGal_N_N was used as forward primer. p-bGal_N_C was used as reverse primer. Annealing was performed at 61 ºC. The amplification step was 90 s long. A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products. Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder Results Na: FINA extraction performed as in protocol. Cl: FINA extraction performed with 10 mM HCl instead of NaOH. L: Cell solution incubated 10 min in 1 mg/ml lysozyme. No washing step in FINA H: Cell solution incubated 10 min at 80 ºC. No washing step in FINA LH: Cell solution incubated 10 min at 80 ºC in 1 mg/ml lysozyme. No washing step in FINA K-: negative control FINA (LB-medium used as sample). P: 1:4 dilution of gRNA PEC #1 after reverse transcription. S: 1:4 dilution of gRNA PEC #1 after FINA extraction for RNA and reverse transcription. KK: Negative control for the cDNA synthesis (water instead of MuLV-RT used) Thursday 31st Intein-Extein-Readout Protocol: E. comp Transformation Participants: Max Observations: E. comp transformation of E. coli Turbo with 1 µl of overnight ligation of 27.8. Platted on Cm plates overnight. RNA extraction from induced Lbu-strain Protocol: Lysis test pipetting scheme Protocol: Phenol-Chlorophorm extraction Protocol: Denaturing PAGE for ssRNA Participants: Dawa Notes: Induced pSB1C3_lbu with IPTG before lysis Two cultures lysed with SDS + Heat only No OD600 measurement Results See Friday, 1st Sept. Friday 1st Intein-Extein-Readout Protocol: Ligation Participants: Max Observations: Plates showed no colonies, as there wasn't enough backbone used. Results Repeated digestion and Ligation from Thursday 31.8 and ligated 1 C-Terminal and 3 N-Terminal fragments respectively. Intein-Extein-Readout Protocol: - Participants: Max Observations: Plates showed no colonies, as there was no SOC step for outgrowth Repeated digestion and Ligation Ligated 1 C-Terminal and 3 N-Terminal fragments into psb1C3 and psb4A5 RNA extraction from induced Lbu-strain (continued) Protocol: Denaturing PAGE for ssRNA Participants: Dawa, Julian Notes: Induced pSB1C3_lbu with IPTG before lysis Two cultures lysed with SDS + Heat only No OD600 measurement Results A B C 1 sample conc ~Nanodrop 260/280 2 1 (non-induced) 2192 1.8 3 2 (non-induced) 2320 1.8 4 3 (induced) 508 1.69 5 4 (induced) 442 1.66 6 1,2 showed precipitation of RNA ->measurement/gel wrong if not vortexed completely A260/280 ration hints to impurities or DNA ->maybe due to too many cells Nukleotide-silica binding with different buffers Protocol: Participants: Julian Notes: Used 10 ul of RNA-solution from Aug 31 RNA-extraction by Dawa per sample Did NOT wash silica particles before with binding buffer (pH) and NOT during procedure with ethanol. Results Both buffers show terrible A260/280 ration, need washing step and need to adjust for acidic silica-solution Saturday 2nd Intein-Extein-Readout Protocol: Chem_Trafo Participants: Julian, Milica, Jorge Observations: Three lysis methods were used: sonifcation, lysozyme digestion and heat lysis in SDS. All Lysozyme digestion: According to protocol. Heat lysis: Sample was incubated in 2% SDS at 75 °C for 15 min. Results See results from "Phenol/Chloroform purification of RNA from in-vitro transcription" from 11.05.17 for gel picture. Nukleotide-silica binding with different buffers Protocol: Participants: Julian Notes: Used 10 ul of RNA-solution from Aug 31 RNA-extraction by Dawa Washed according to protocol Results Acetate-buffer results acceptable now, GuSCN-buffer still a problem.GuSCN probably too difficult clean up, needs too many washing steps. Monday 4th Intein-Extein-Readout Protocol: E. comp Transformation Participants: Max Observations: Plates showed no colonies as there was no SOC step. Retransformation of Ligation from Friday into E.Coli Turbo e. comp. Total RNA extraction of E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with Norgen kit Protocol: Total RNA Purification with Norgen Kit Participants: Jorge, Dawafuti Observations: Stored at -80 ºC as TRNA Kit #1-4 Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT Protocol: Protocol Participants: Jorge, Dawafuti Observations Stored at -80 ºC as TRNA PC #1-4. Results See results Urea-SDS-PAGE from 05.09.17. Nukleotide-silica binding with different buffers Protocol: Participants: Julian Notes: Did NOT incubate silica with acetate buffer for a day, prepared it fresh Used 10 ul of RNA-solution from Aug 31 RNA-extraction by Dawa Washed according to protocol, but did NOT wait a day for silica-buffer interaction with Acetate-buffer Results Terrible yield, need buffer-silica incubation in protocol. Tuesday 5th DNA Amplification Test using Recombinase Polymerase Amplification Protocol: Recombinase Polymerase Amplification Participants: Sven Observations: DNA Amplification with RPA was tested using the provided Control Reaction by TwistDx and our BioBrick His6-TEV plasmid incubation times of 40 minutes for Control and Sample 1 and 60 minutes for sample 2. Differences due to incubation time was tested. RPA according to protocol PCR cleanup according to protocol. Results Positive Control worked. RPA of TEV could not produce the whole (~1300 bp) construct but would produce amplicons of shorter size (100-500 bp) which was anticipated since TwistDx protocol says optimal amplicon length is 500 bp. Full length double-stranded DNA could consequently not be formed. Intein-Extein-Readout Protocol: Agarose-Gel Participants: Max Observations: Ran a small 1.75% TAE-Agarose Gel at 150 V for 50 minutes and post stained with Sybr Gold Overnight Ligation of Intein-Extein-Construct Protocol: Ligation of DNA-Fragments Participants: Max Observations: Overnight ligation in ice bath with psb1C3 and psb4A5 as backbones and a 3:1 molar excess of insert 200 ng backbone Preparation of chemical competent Turbo cells Protocol: Preparation of chemical competent cells Participants: Max Observations: Plated one aliquot as negative controll Results No colonies on plates, therefore usefull competent cells. Invitro Transcription IC3a RNA Protocol: In vitro transcription Participants: Florian Observations: Results See results of DNAseI digestion and PCE from 06.09.17 Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT (continued) Protocol: Denaturing PAGE for ssRNA Participants: Dawa, Jorge Notes: Ran urea gel Results DNA Amplification Test using Recombinase Polymerase Amplification Protocol: Recombinase Polymerase Amplification Participants: Sven Observations: DNA Amplification with RPA was tested using the provided Control Reaction by TwistDx and our BioBrick His6-TEV plasmid incubation times of 40 minutes for Control and Sample 1 and 60 minutes for sample 2. Differences due to incubation time was tested. RPA according to protocol PCR cleanup according to protocol. Results Positive Control worked. RPA of TEV could not produce the whole (~1300 bp) construct but would produce amplicons of shorter size (100-500 bp) which was anticipated since TwistDx protocol says optimal amplicon length is 500 bp. Full length double-stranded DNA could consequently not be formed. Wednesday 6th RNaseAlert Experiment with E.coli RNA with different concentrations of target E.coli RNA Protocol: RNase activity Participants: Dawa, Jorge, Igor Notes: Run1: Amount of RNA: 1 nm Cas13a, 10 nm crRNA Run2: Amount of RNA: 1 nm Cas13a, 10 nm crRNA img src="https://static.igem.org/mediawiki/2017/b/be/T--Munich--pic--platereader_pipScheme_6_09_Ecoli.png" > Intein-Extein-Readout Protocol: Chem and electro Transformation Participants: Max Observations: Transformation into chem and e. competent E.coli Turbo and DH5 alpha cells. Results No colonies in the evening, therefore the plates were left at room temperature overnight. DNAse I digest and PCE of invitro transcribed IC3a RNA Protocol: Restriction digest,Phenol-chlorophorm purification of DNA Participants: Florian Observations: Results 6 M UREA-PAGE 15% of IC3a RNA Thursday 7th RNA Intein-Extein-Readout Protocol: Colony PCR Participants: Max Observations: Picked 128 colonies from plates of 06.09.17 4 strip tubes of each construct( psB1C3_C; psB1c3_N; psB4A5_c; psB4A5_N) Colony PCR with OneTaq Polymerase Samples loaded onto monster Gel (Samples order is interlaced) Results First try of Cas13a digestion of IC3a/C3a dimer over 1 hour Protocol: RNAse activity protocol Participants: Florian Observations: Incubation for dimerization at room temperature for 1 h. Afterwards 1 hour digestion with Cas13a. Results No real difference visible after digestion for 1 h. Friday 8th Test heat-only lysis (1,3 and 7 minutes)) Protocol: Phenol-Chlorophorm extraction Participants: Julian Notes: Diluted E. coli culture to 108 cells/ml, used 300 uL as sample Heat-only at 93 °C for 15 min, diluted wiht tap water to simulate actual usage scenario (saliva etc..) Results Hardly any RNA detectable (concentration < 10 ug/ml). Abysmal 260/280-ratio -> Heat-only lysis is too inefficient for that low cell counts, use 109 next time. Monday 11th Intein-Extein-Readout Protocol: - Participants: Max Observations: Precultures of 16 colonies from plates of 06.09.17. Precultures in 5 ml LB with corresponding antibiotic Second try of Cas13a digestion of IC3a/C3a dimer for several hours and overnight Protocol: RNAse activity protocol Participants: Florian Observations: Results Tuesday 12th Intein-Extein-Readout Protocol: Mini-Prep with Qiagen Kit and Sequencing Sample preparation Participants: Max Observations: Mini Prep of precultures from 11.09.17 Send all 16 plasmids for sequencing with primer VR Results psB4A5_C-Terminal 1-7 and 3-4 were positive. Wednesday 13th Intein-Extein-Readout Protocol: Restriction digest and PCR protocol with Q5 polymerase Participants: Max Observations: Send Samples psB4A5_C-Terminal 1-7 and 3-4 for sequencing with primer VF New digestion of unamplified N-Terminal g-block (200 ng in 50 µl with EcoRI-HF and SpeI-HF) PCR of psB1C3 with psb1A3_fw and psb1A3_rev , to get an opened backbone (Q5 polymerase protocol with 66 °C as annealing temerature and 1 minute elongation time) Results PsB4A5_C terminal 1-7 was sequenced completly and used for further cloning steps. Heat-lysis with E. coli Protocol: Phenol-Chlorophorm extraction Participants: Julian Notes: Lysis at 90°C, used 300 uL of E. coli suspension diluted to 2*109 cells/ml SDS-Lysis as reference was impossible with this cell count, interphase during extraction too broad Results Extraction-yield is down 5- to 10- fold compared to SDS (calculated from earlier SDS-lysis results with 109 (not 2*109) RPA stability test and Reaction Temperature Screening on paper Protocol: Recombinase Polymerase Amplification on Paper Participants: Sven Observations: Preparation of stability assay of RPA on paper. Lyophilisation and storage. RPA on paper according to protocol Thursday 14th RPA stability test and Reaction Temperature Screening on paper Protocol: Recombinase Polymerase Amplification on Paper Participants: Sven Observations: Preparation of stability assay of RPA on paper. Lyophilisation and storage. RPA on paper according to protocol Intein-Extein-Readout Protocol: Restriction digest, Ligation of DNA fragments, Gibbson-Assembly and E and chem Trafo Participants: Max Observations: New digestion of unamplified N-Terminal gBlock and psB1C3 with SpeI-HF and EcoRI-Hf Dephosphorylation with Antarctic Phosphatase Ligation of digested sequences overnight with T4 DNA Ligase Gibbson-Assembly of psB1C3with N-Term fragment Transformation of chem and e. competent Turbo cells Friday 15th RPA stability test and Reaction Temperature Screening on paper Protocol: Recombinase Polymerase Amplification on Paper Participants: Sven Observations: Stability of RPA on paper was tested using His6-TEV plasmid after 0, 2 and 24 hours post-lyophilisation. Reaction temperature was tested at 37 °C as suggested by TwistDx and room temperature. Reaction was positively controlled using non-lyophilised reaction mixture. RPA according to protocol PCR cleanup according to protocol. Results Lyophilisation of RPA on paper worked. Directly after lyophilisation, the reaction efficiency seems to be similar to fresh RPA reaction mixture. Lyophilisation of RPA on paper worked. However, activity loss is observed already after 2 hours and after 24 hours, almost no activity is observable anymore. Intein-Extein-Readout Protocol: Colony PCR Participants: Max Observations: Picked 32 colonies from plates of 14.09.17 Screened with Colony PCR Gel 200 V 2 % Agarose 30 minutes Results No positive colononies in the Col PCR. Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT Protocol: Phenol/Chloroform purification of RNA Participants: Jorge Observations: Two samples stored at -80 ºC labeled TRNA Lsh #1 15.09.17 and TRNA Lsh #2 15.09.17 Results Monday 18th RPA stability test on paper Protocol: Recombinase Polymerase Amplification on Paper Participants: Sven Observations: Preparation of temperature and air-sensitivity assay of RPA on paper. RPA on paper according to protocol. Results Lyophilisation of RPA on paper. Storage with and without Parafilm enclosure at 4 °C or room temperature. Intein-Extein-Readout Protocol: Q5-PCR and Restriction digestion and Col-PCR and e and chem comp Transformation Participants: Max Observations: New PCR of backbone with psB1A3-fw and psB1A3-rev. Sample was subsequently DpnI digested and purified Col-Pcr of 16 non green colonies from plates from thursday 14.9.17 Transformation of cells with ON Ligation from Friday 15.9.17 into E.coli DH5 alpha and E.coli Turbo Tuesday 19th RNA extraction from 5ml overnight culture of B. subtilis Protocol: Phenol-chlorophorm extraction RNA Participants: Dawa Notes: used heat lysis at 90 °C for 15 mins Coupling In-Vitro Transcription to RPA Protocol: Coupling RPA to In-Vitro Transcription Participants: Sven Observations: Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. The construct as template used was His6-TEV plasmid. RPA-Tx according to protocol. Phenol-Chloroform-Extraction according to protocol. Results In-Vitro Transcription could not be shown. RNA was detected in the samples in which no T7 RNA Polymerase was added while there are no bands in the samples that had T7 RNA Polymerase in them. Though unlikely, a labelling error might have occured. RPA stability test on paper Protocol: Recombinase Polymerase Amplification on Paper Participants: Sven Observations: Stability of RPA on paper was tested using His6-TEV plasmid after 24 hours post-lyophilisation. Storage conditions were altered. Storage at room temperature and at 4 °C were tested. Also, duplicates were done at these temperatures, one sample being always enclosed with Parafilm to avoid air exposure. RPA on Paper according to protocol PCR cleanup according to protocol. Results RPA activity clearly showed that storage temperature did not have a significant effect on the integrity of the RPA reaction mixture. Exposure to air, however, was identified as the bottleneck for RPA stability. The RPA samples that were protected from exposure to air showed significantly higher Intein-Extein-Readout and Biobrick Recloning Protocol: Gibbson-Assembly and Ligation Participants: Max, Rob Observations: Gel of Col-PCR from 18.09.17 Clean-up from backbone PCR and digest from 18.09.17 followed by Gibbsonassembly with N-terminal insert. Started the Recloning of the Biobricks by PCR with phosphorylated primers and T4 Ligation and transformed into Turbo chem competent cells. Results Sample 1-3 and 2-1 had the correct length, and therefore precultures of these colonies were started overnight. IC3a invitro transcription Protocol: In vitro transcription Participants: Florian Observations: Results See results of DNaseI digestion and PCE from 20.09.17. Heat-lysis with B. subtilis Protocol: Phenol-Chlorophorm extraction Participants: Julian Notes: 2x SDS-Heat lysis (protocol), 2x heat-only at 90 °C for 20 min Results SDS-lysis works a lot worse for gram+ bacteria, Heat-only now better/same than SDS-lysis (more incubation time...) A260/280 ratio relatively bad, probably a lot of cell-wall impurities Heat lysis and isothermal PCR: temperature dependence Protocol: RPA-pcr Participants: Julian, Sven Notes: Diluted E. coli (DH5a-pSB1C3-Tev-008-His) suspension to 106 cells / ml and heat-lysed in 50 uL aliquots Lysis-temperature ranged from 65 °C to 90 °C in 5 °C steps Added 2 uL of lysed sample to 10 uL RPA-solution and TEV-protease primers Results Heat differences in this range and this cell concentration have only minor effect. Best lysis seems to be around 80 °C, which is consistens with literature. Wednesday 20th Gel analysis of B.subtilis RNA samples Protocol: Denaturating Urea PAGE Participants: Dawa Notes: 8% gel Results B. subtilis-PCE 190 ng/ul, B.subtilis heat/lysis-378 ng/ul Intein-Extein-Readout Protocol: Miniprep with Qiagen Kit Participants: Max Observations: Minipreped overnight cultures from 1-3 and 2-1. Send samples for sequencing with VF and VR. Results N-Terminal sample 1-3 was positive. Biobrick Recloning Protocol: Col PCR and preparation of e comp. cells Participants: Max Observations: Colony PCR of 36 colonies from each of the 3 constructs His-Sumo-Lwa (Lwa(l)), Lwa(lwa(k) and His-Sumo-Lbu each in psB1C3. Precultures of 4 colonies from Lwa(l) and Lwa(k) Results DNAse I digest and Phenol-Chloroform-Precipitation Protocol: Restriction digest,Phenol-chlorophorm purification of RNA Participants: Florian Observations: Results 6 M UREA-PAGE of invitro transcribed and pooled IC3a. Quantitative gel analysis calculated a concentration of 35 µM. Thurdsay 21st Intein-Extein-Readout Protocol: Restriction digest, Gibbson-Assembly and Transformation Participants: Max Observations: Digestion of 1 µg of each backbone (psb1c3_N-term_uni and psb4A5_C-Term_uni with BBS1-HF to open them for Gibbson-Assembly Gibbson-Assembly of Col_PCR fragments from 03.08.2017 1:3 backbone to insert concentration 30 minutes at 50 °C Transformation of E.coli Turbo e and chem competent cells according to protocol Preculture for Cryostock from psB1C3_Split_uni_N_term 1-3 Results Biobrick Recloning Protocol: Col-PCR and Sample Sequencing Participants: Max Observations: Send samples for sequencing Col_PCR of 4 samples from Lbu plate (all negative) Wednesday 20th RNase Alert Experiment with Bacillus subtilis using varying concentrations of target RNA Protocol: RNase activity Participants: Dawa Notes: Pippeting scheme: Thursday 21th B. subtilis RNA extraction using lysozyme Protocol: Phenol-chlorophorm RNA extraction Participants: Dawa Monday 25th crRNA in vitro transcription (for B.subtilis, Norovirus. Q5 beta, Hepatitis) Protocol: In vitro Transcription Participants: Dawa Intein-Extein-Readout Protocol: Restriction digest, PCR Cleanup Kit, Gibbson Assembly and Transformation Participants: Max Observations: Redigest of 1 µg of each backbone with BBS1 and dephoshorilation according to protocol Cleanup with cleanup kit Gibbson assembly with 100 µg backbone and 3:1 excess of insert ( calculated based on length ratio) Inserts 2 and 3 from PCR of 05.08.17 Transformation into E.coli Turbo e. comp COlPCR of plates from Thursday. 21.9.17=> 36 samples Gel at 1 % 45 mins 150 V Biobrick Recloning Protocol: Total Participants: Max Observations: COlPCR and Gel of samples from trafo of Friday 22.09.17 => 30 samples (all non green) psB1C3_LBU Gel at 1 % 45 mins 150 V Results All negative. Tuesday 26th crRNA quantification, Urea gel run Protocol: Denaturating Urea Page Participants: Dawa, Erika Notes: 2 samples each from B. subtilis, Norovirus, Q5 beta and HCV(first diluted 1:5 and second undiluted) Intein-Extein-Readout Protocol: ColPCR, Agarosegel Participants: Max, Milica Observations: ColPCR of 12 colonies from N-term plate from 25.9.17 Gel at 1 % 30 mins 150 V Results Samples 1-8 and 2-1 of the N-Terminus looked good => preculture for sequencing. Biobrick Recloning Protocol: Total Participants: Max Observations: COlPCR and Gel of samples from trafo of Friday 22.09.17 => 30 samples (all non green) psB1C3_Lwa_kurz Gel at 1 % 30 mins 150 V Results All negative. Wednesday 27th Intein-Extein-Readout Protocol: Minipreparation and Sample Sequencing Participants: Max Observations: Miniprep of psB1C3_Split_N_term_bGal 1-8 and 2-1 Send Minipreps for sequencing with primer VF and VR Results Both sequences positive. Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT Protocol: Phenol/Chloroform purification of RNA Participants: Jorge Observations: Four samples stored at -80 ºC labeled TRNA Lsh #1 27.09.17 and TRNA Lsh #2 27.09.17, TRNA Lsh #3 27.09.17, TRNA Lsh #4 27.09.17. Results Thursday 28th Agarose and Urea gel run with RNA Protocol: PAGE, Urea PAGE Participants: Dawa, Erika Results Agarose Gel didn’t work – The samples seems to be degraded in agarose gel , may be due to contamination of the gel chamber and other materials with Rnase. Biobrick Recloning Protocol: ColPCR, ON culture Participants: Max Observations: ColPCR from samples of 22.09.17 32x Lwa short Overnight cultures from colonies 1-8 and 2-3 started Results RNA/DNA hybrid dimerization with Cy5 DNA-oligo C3a Protocol: Participants: Florian Observations: Results Native PAGE 20%, 12.5 mM MgCl2 C3a too high concentrated (Red bands). 5 µM instead of 500 nM. Better results with band shift see 29-09-2017. Screen of LBU activity on RNA/DNA dimer Protocol: RNAse activity protocol Participants: Florian Observations: Results Native PAGE 20%, 12.5 mM MgCl2 No visible shift of activator DNA oligo with addition of RNA under the use of Cy5 modified DNA activator oligo. Results are bad because of too high C3A DNA oligo concentration. (C3a = red bands). Friday 29th RNaseAlert experiment with extracted RNA targets from E.coli and in vitro transcribed RNAs. Protocol: RNase activity Participants: Dawa, Erika Notes: Pipetting scheme: Results The results for E.coli look good. Excel table saved in lrz as: 2017_09_29_rnase alert_ecolitarget_invitrotarget.xlsx Biobrick Recloning Protocol: Mini-Prepatation Participants: Max Observations: Mini prep of 1-8 and 2-3. Lwa short send for sequencing with VF and VR Results Lwa long positive. DNA and RNA hybrid of 3C-A and IC3-A Protocol: Participants: Florian Observations: Results Native PAGE 20%, 12.5 mM MgCl2 Visible small up shift of RNA/DNA dimer versus samples, which only contain the DNA oligo by itself. (DNA oligo C3a marked with Cy5 visible as red bands.) FINA extraction for RNA with purified total RNA (TRNA Lsh #2-4 from 27.09.17) Protocol: FINA Extraction for RNA Participants: Jorge Observations: FINA was performed with 25 ul sample instead of 50 ul. The extraction for TRNA #2 was performed as in protocol. Before the FINA extraction, TRNA #3 was digested with DNase I according to DNase I Reaction Protocol After the FINA extraction, the two eluate from TRNA #4 were digested with DNase I according to DNase I Reaction Protocol First strand cDNA synthesis from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT FINA-RNA eluate Protocol: First strand cDNA synthesis Participants: Jorge Observations pseq-Lsh-06rev was used as primer. The eluates from the FINA extractions for RNA from 29.09.17 were used as samples. From each pair of samples, one was used as a negative control (nuclease free water instead of MuLV-RT). For all reactions, 6 ul sample were pipetted. Q5-PCR for Lsh-fragment in E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with cDNA samples from 29.09.17 Protocol: PCR amplification with Q5 Master Mix Participants: Jorge Observations: pseq-Lsh-05fwd was used as forward primer. pseq-Lsh-06rev was used as reverse primer. Annealing was performed at 58 ºC. The amplification step was 110 s long. A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products. Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder Results N: FINA extraction performed as in protocol. NK: FINA extraction as in protocol. No RT in cDNA synthesis (negative control). P: Digestion of DNA before FINA extraction. PK: Digestion of DNA before FINA extraction. No RT in cDNA synthesis (negative control). A: Digestion of DNA after FINA extraction. AK: Digestion of DNA after FINA extraction. No RT in cDNA synthesis (negative control). Monday 2nd Phenol chloroform purification of crRNAs of B. subtilis, NoroV, HCV and Q5 beta Protocol: Phenol-chloroform extraction RNA Participants: Dawa Live Invitro Transcription test with LBU and Inhibitor-RNA/Activator-DNA dimer with different T7 concentration Protocol: In vitro transcription Participants: Florian Observations: Forgot to add C3ds with single strand overhang, which is needed for activation of invitro transcription after binding of DNA oligo C3a (activator of C3ds). Results Plate reader data showed no activity for all samples, as expected without DNA template to transcribe. Tuesday 3rd various Rnase alert experiments Protocol: RNase activity Participants: Dawa Notes: with Noro Virus (10 ul total volume used for the experiment) with Q5 beta (10 ul total volume used for the experiment) with Bacillus ( 10 ul total volume used for the experiment) and with HCV and E.coli RNA (FINA method) samples (10 ul total volume used for the experiment) Pipetting Scheme Norovirus and Q5 beta: Pipetting Scheme JVC and B. subtilis: Pipetting Scheme E.coli RNA (FINA method): Results Bacillus, Noro virus and HCV seeem to work. However Q5 beta needs to be repeated and also HCV could be repeated! FINA extraction of RNA from E. coli with and without filters Protocol: FINA RNA extraction Participants: Jorge Results Works, Target-sequence with PCR detected. Live Invitro Transcription test with LBU and Inhibitor-RNA/Activator-DNA dimer with different T7 concentration Protocol: In vitro transcription Participants: Florian Observations: Results No Invitro transcription of RNA at any T7-Polymerase concentration. LBU does either not set activator DNA free or does not work at all. Same result as on the gel. RNAse H shows low level fluorescence over the whole experiment, which can not be related to an invitro transcription. Wednesday 4th Bacillus RNA extraction Protocol: RNase activity Participants: Erika, Dawa Notes: Resuspension Buffer (20 ml): Sucrose (0.3 M) -> 2.1 g Sodium Acetate (10 mM) -> 16.4 mg Adjust pH to 4.2 Stored at 4°C Biobrick Recloning Protocol: - Participants: Max Observations: Send Lwa short colony 2-3 for sequencing with pseqLwa primer Results Full read with no mutations. Thursday 5th RNaseAlert Protocol: RNase activity Participants: Dawa, Rob, Erika Notes: RNaseAlert reaction mix ( in glass fiber filter paper) lyophilisation. Introduction and first trial Intein-Extein-Readout Protocol: Transformation of e. comp cells Participants: Max Observations: Repeated Gibbson-Assembly for Lbu with 4:1 ratio Transformation into E.coli Turbo Results Empty plates => need to repeat PCR. In-vitro transcription of AuNP linkers U0, U5, U10, U15 Protocol: in vitro transcription Participants: Rob Friday 6th RNA extraction from Bacillus overnight culture Protocol: Phenol-chlorophorm extraction Participants: Erika Notes: following SIGMA Genosys Protocol from Step 1 to 7. Then, followed Lab´s protocol steps. RNA sample stored at -80°C and needs to be quantified. PCE and gel analysis of of AuNP linkers U0, U5, U10, U15 and new in vitro transcription Protocol: phenol-chlorophorm purification of RNA Protocol: Urea gel for RNA detection Protocol: in vitro transcription Participants: Rob Observations: There was no RNA visible. The in-vitro transcription was repeated. Results Saturday 7th SybrGreen II sensitivity test Protocol: Participants: Florian Observations: Results 1:10.000 seems to be the right dilution of SybrGreen II dye for a 15 µl Sample. PCE and gel analysis of of AuNP linkers U0, U5, U10, U15 Protocol: phenol-chlorophorm purification of RNA Protocol: Urea gel for RNA detection Protocol: in vitro transcription Participants: Rob Observations: There was no RNA visible. The in-vitro transcription was repeated with a new batch of T7 RNA polymerase. Sunday 8th IC3A invitro transcription Protocol: Invitro transcription Participants: Florian Observations: Results See results of Digestion and PCE from 09.10.17. T7 polymerase sensitivity test Protocol: Invitro transcription Participants: Florian Observations: Results Higher concentrations of T7-polymerase causes the maximal concentration of RNA to be reached faster. PCE and gel analysis of of AuNP linkers U0, U5, U10, U15 Protocol: phenol-chlorophorm purification of RNA Protocol: Urea gel for RNA detection Protocol: AuNP preparation Participants: Rob Observations: The IVTX was successful. 3-day incubation of AuNP from stock was started. <\li> Results Monday 9th Quantification of viral RNAs and Bacillus RNA extracted from last week Protocol: RNA urea gel Participants: Dawa Lbu-Recloning with Gibbson-Assembly Protocol: PCR,Gibbson and Trafo in chem and e comp Participants: Max Observations: PCR for Gibbson with Biobrick fwd and rev primer. Lbu Gibbson Assembly with 3x excess of insert. Trafo into E. coli chem and e comp. Results No colonies DNAse I digest and PCE of 4 pooled invitro transcriptions of IC3a Protocol: Restriction digest,Phenol-chlorophorm purification of RNA Participants: Florian Observations: Results 6 M UREA-PAGE 15% of IC3a RNA Tuesday 10th Intein-Extein-Readout Protocol: PCR and Gibbson Assembly Participants: Max Observations: PCR of Lbu and psB1C3 as backbone Gibbson Assembly with 5:1 excess of insert Results Heat lysis and isothermal PCR: cell concentration dependence s Protocol: RPA-pcr Participants: Julian, Sven Notes: Diluted E. coli (DH5a-pSB1C3-Tev-008-His) suspension in a range of 106 to 102 cells / ml and heat-lysed in 50 uL aliquots Added 2 uL of lysed sample to 10 uL RPA-solution and TEV-protease primers Results Bands visible down to a dillution of 5*104 or 104 cells/ml, which corresponds to an average of 100 or 20 cells' DNA per PCR Wednesday 11th Plate reader experiments Protocol: RNase activity Participants: Dawa, Jorge Notes: with Noro virus samples ( worked!) with Bacillus ( seems to have Rnase contamination) with the paper strips (glass fiber filter paper) 1.8 ul reaction mix pipetted with different amounts of invitro transcribed RNAs. with E.coli RNA samples( heat lysed)- ( seems to have RNase contamination) Pipetting Scheme 1: Pipetting Scheme 2: Coupling In-Vitro Transcription to RPA in Bulk and on Paper Protocol: Coupling RPA to In-Vitro Transcription Participants: Sven Observations: Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. The construct as template used was RNA Amp G-Block 200 bp double-stranded DNA using VF and VR as primers. RPA-Tx and RPA-Tx on Paper according to protocol. G-Block cloning of RNA-Amplification test plasmid via Restriction Cloning and Gibson Assembly Protocol: G-Block Cloning Participants: Sven Observations: Preparation: Solubilisation of G-Block to reach 10 ng/ul concentration. PCR reaction using VF and VR as primers resulted in 50 ul of a 24 ng/ul solution. Gibson Assembly: PCR reaction using pSB1A3_fw and pSB1A3_rev for backbone amplification of pSB1C3-GFP yielded 54 ng/ul. Ratio for Gibson Assembly: 0.375 pmol Insert : 0.125 pmol backbone. Incubation for 15 minutes at 50 °C. Restriction Cloning: Restriction of backbone and insert using EcoRI-HF and SpeI-HF in NEB Cutsmart Buffer and heat inactivation. Subsequent dephosphorylation of backbone using Antartic Phosphatase. T4 DNA Ligation with a ratio of 0.02 pmol backbone : 0.06 pmol insert. Transformation of Ligation and Gibson Assembly into chemocompetent and electrocompetent Turbo cells. Plate-out on chloramphenicol agar plates. Results The investigation of the agar plates the next day using UV-light revealed that the only plasmid that was taken up by the Turbo cells was the initial pSB1C3-GFP plasmid. No positive inserts could be identified. Biobrick Recloning Protocol: Transformation of chem comp cells Participants: Max Observations: Transformation of Turbo cells with Gibbson of 10.10. Dye Test for live imaging of invitro transcription (1st row Controls (with SybrGold partially), 2nd row SybrGold dilution series, 3rd row SybrGreen I dilution series, 4th row EvaGreen dilution series) Protocol: invitro transcription Participants: Florian Observations: Results 6 M UREA-PAGE 15% of IC3a RNA Mistake made: Added T7 Polymerase to Control 2. Was left out in Graph. Only Eva Green seems to not influence the invitro transcription, because it was especially designed for live imaging with ongoing transcription reactions. Cas13a activity assay Protocol: Cas13a activity assay Participants: Igor Observations: Results Preparation and DNA-conjugation of of new AuNP Protocol: AuNP preparation Protocol: AuNP-DNA conjugation Participants: Rob Observations: Preparation of AuNP was successfully completed. DNA-conjugation with "AuNP 5' primer" and "AuNP 3' primer" <\li> Results See results from "conjugation test" Thursday 12th Plate reader experiment with paper strips blocked ovenight with BSA Protocol: RNase activity Participants: Dawa Notes: 1.8 ul of reaction mix pipetted in each paper with different amounts of in vitro target RNA. The overnight blocked paper strips were rinsed with nuclease free water and then dried in the oven at 70 degree for 30 mins and then used for the experiments. Pipetting scheme: Results Seems to work! Needs to be repeated for the confirmation of results. UREA-PAGE of Coupling In-Vitro Transcription to RPA in Bulk and on Paper Protocol: Coupling RPA to In-Vitro Transcription Participants: Sven Observations: Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. The construct as template used was RNA Amp G-Block 200 bp double-stranded DNA using VF and VR as primers. RPA-Tx and RPA-Tx on Paper according to protocol. UREA-PAGE Gel of Samples extracted via Phenol-Choloform-Extraction; both according to protocol. Results T7 RNA-Polymerase was, by accident, also added to the negative control of the RPA-Tx reaction on Paper. First, one can see that the gel was overloaded. Also, it seems like there is additional bands at the expected size of around 150 bp. Questionable is that this seems also to be the case for the negative Control of the RPA-Tx reaction in Bulk. The experiment on paper, however, looks quite promising since it seems like there are distinct bands at the expected approximate size of the RNA transcript. Cas13a cleavage assay will provide evidence if these bands actually consist of the desired target RNA. Intein-Extein-Readout Protocol: Transformation of e. comp cells Participants: Max Observations: Transformation of BL21 with psB4A5 C-Term and psB1C3 N-Term Results No colonies for psB4A5 C-Term. Biobrick Recloning Protocol: Col-PCR for Biobrick Recloning Participants: Max Observations: Col PCR of 6x N-Term in psB1C3, 16x C-Term in psB1C3, 14x Lbu in psB1C3 Results All negative. Eva Green Sensitivity Test with RNA background Protocol: Participants: Florian Observations: AuNP linkage asssay with DNA-linker Protocol: AuNP linkage Participants: Rob Observations: 1x Buffer was used first, which resulted in no aggregation. Increasing the concentration by adding buffer to a final concentration of 2x afterwards resulted in aggregation and thus was set as the standard. Friday 13th Plate reader experiment with paper strips blocked ovenight with BSA Protocol: RNase activity Participants: Rob Notes: 1.8 ul of reaction mix pipetted in each paper with different amounts of in vitro target RNA. The overnight blocked paper strips were rinsed with nuclease free water and then dried in the oven at 70 degree for 30 mins and then used for the experiments. (Results seem to work!) Pipetting scheme: Biobrick Recloning Protocol: PCR with Q5-Polymerase Participants: Max Observations: Repeated PCR of N and C-term part for Recloning AuNP linkage asssay with DNA-linker and varying buffer concentrations Protocol: AuNP linkage Participants: Rob Observations: 2x, 4x, and 6x buffer was used, which resulted in unspecific aggregation from 4x to 6x buffer, thus 2x was kept as standard. Saturday 14th Intein-Extein-Readout Protocol: Restriction digest Gibbson-Assembly and Transformation Participants: Max Observations: DpnI digest of PCR from 31.10.17 Clean-up with PCR purification kit Gibbson-Assembly with 3:1 excess Transformation into E.coli Turbo chem comp. Sunday 15th RNA extraction of E. coli and Bacillus + plate reader experiment Protocol: RNase activity Participants: Dawa, Jorge, Erika Notes: HCV assay and Q5beta in Fluorostar and Clariostar for 1 hour each (HCV assay worked!) RNA urea gel for RNA extracted from E.coli and Bacillus Plate reader experiment of RNA extracted from the E.coli in Fluorostar for 2 hours Pipetting scheme: Intein-Extein-Readout Protocol: Transformation of e. comp cells Participants: Max Observations: Repeated Transformation of Bl21 star with psB4A5-Split-Intein-C-Term Results No colonies.=> Transformation in other expression strains Monday 16th Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative controls. Plus assay with cas13a with TDPs and assay with RPA paper samples. Protocol: RNase activity Participants: Dawa Pipetting scheme: Intein-Extein-Readout Protocol: Transformation of e. comp cells Participants: Max Observations: Transformed all available Bl21 strains with psB4A5-C-Term Strains were: Bl21 pLYs; BL21 star; BL21 DE3, BL21 RIPL, Rosetta and E.coli Turbo as pos control Results No colonies for any BL21 strain. The Turbo cells grew fine. => Expression seems to be toxic. New DNA-conjugation and AuNP linkage asssay with RNA-linker and DNA-linker Protocol: AuNP linkage Protocol: AuNP-DNA conjugation Participants: Rob Observations: New AuNP were DNA-conjugated Water was used instead of linker-RNA. Tuesday 17th Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative controls. Plus assay with cas13a with TDPs and assay with RPA paper samples. Protocol: RNase activity Participants: Dawa Pipetting Scheme B. subtilis: Pipetting Scheme RPA paper: Intein-Extein-Readout Protocol: - Participants: Max Observations: Midipreparation of psB4A5-C-Term with Macherey-Nagel Xtra Midi Preparation done according to protocol Plasmid was intended for In vitro Expression Results In vitro expression was not done, due to lack of time. Biobrick Recloning Protocol: Q5-PCR, Gibbson-Assembly and Transformation Participants: Max Observations: PCR of all GFP degradation tag library variants with Biobrick sequence binding primers, for transfer from psB1A3 to psB1C3. Gibbson-Assembly and Transformation according to protocol Results AuNP linkage asssay with RNA-linker and DNA-linker Protocol: AuNP linkage Participants: Rob Observations: The experiment from the day before resulted in unspecific aggregation in the negative control. To have comparable samples and negative control for the cleavage assay, the experiment was repeated with a mock-DNA in the negative control. repeated with mock-RNA as negative control. A possible explanation for this is that negatively charched, non-bound RNA in the solution increases repulsion of AuNP. Wednesday 18th Biobrick Recloning Protocol: Overnight culture Participants: Max Observations: Picked 2 green colonies from each plate for overnight culture observation of AuNP linkage asssay with RNA-linker and DNA-linker Protocol: AuNP linkage Participants: Rob Observations: After cooling over night and spinning down for 1 min, just minor aggregation was achieved and samples were kept at 4°C for 2 days to allow for potential further aggregation. Thursday 19th G-Block cloning of RNA-Amplification test plasmid via Restriction Cloning and Gibson Assembly Protocol: G-Block Cloning Participants: Sven Observations: Preparation: PCR reaction of G-Block using VF and VR as primers resulted in 50 ul of a 27 ng/ul solution. Gibson Assembly: PCR reaction using pSB1A3_fw and pSB1A3_rev for backbone amplification of pSB1C3-GFP yielded 52 ng/ul. Ratio for Gibson Assembly: 0.375 pmol Insert : 0.125 pmol backbone. Incubation for 15 minutes at 50 °C. Restriction Cloning: Restriction of backbone and insert using EcoRI-HF and SpeI-HF in NEB Cutsmart Buffer and heat inactivation. Simultanous dephosphorylation of backbone using Antartic Phosphatase. T4 DNA Ligation with a ratio of 0.02 pmol backbone : 0.06 pmol insert. Transformation of Ligation and Gibson Assembly into chemocompetent and electrocompetent Turbo cells. Plate-out on chloramphenicol agar plates. Results The investigation of the agar plates the next day using UV-light showed that for the Gibson Assembly, positive clones were visible. Colony PCR was performed and showed inserts for several plasmids. Overnight cultures were inocculated for sequencing. Biobrick Recloning Protocol: Sequencing Participants: Max Observations: Send samples from degradation tag library for sequencing Results pdt2-E 2 was positive Friday 20th G-Block cloning of RNA-Amplification test plasmid Protocol: G-Block Cloning Participants: Sven Observations: Mini-prep of previously inocculated overnight cultures and sent to sequencing. Results The investigation of the agar plates the next day using UV-light showed that for the Gibson Assembly, positive clones were visible. In vitro transcription of crRNA Lbu, crRNA Lsh and target RNA at 37 ºC overnight Protocol: In vitro Transcription Participants: Florian See results of digestion and PEC from 21.10.17 observation of AuNP linkage asssay with RNA-linker and DNA-linker Protocol: AuNP linkage Participants: Rob Observations: The 2-day incubation did not result in higher aggregation, so a new over-night linkage was started. <\li> After cooling over night and spinning down for 1 min, just minor aggregation was achieved and samples were kept at 4°C for 3 days over the weekend to allow for aggregat Saturday 21st DNAse I digest and PCE of the RNA crRNA from Lbu and Lsh Protocol: Restriction digest,Phenol/Chlorophorm purification of RNA Participants: Florian Observations: Results In vitro transcription of crRNA Lwa at 37 C overnight Protocol: In vitro Transcription Participants: Florian See results of digestion and PEC from 23.10.17 Sunday 22nd RPA on PDMS Chip Protocol: RPA on Paper Participants: Sven Observations: Since the PDMS chip was leaky, no real sample could be extracted from it. Results Positive Controls as well as colony-PCR using RPA showed clear bands on a 2%-agarose gel. The RPA performed on Chip, however, was not showing any bands though Nanodrop concentration showed 27 ng/ul in the sample. observation of AuNP linkage asssay with RNA-linker and DNA-linker Protocol: AuNP linkage Participants: Rob Observations: After cooling over night and spinning down for 10 min, aggregates could be observed more clearly. DNAse I digest and PCE of the RNA crRNA from Lwa Protocol: Restriction digest,Phenol/Chlorophorm purification of RNA Participants: Dawafuti Observations: Results Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative controls with Lsh Cas13a Protocol: RNase activity Participants: Dawafuti Observations: Results No activity of Lsh Cas13a visible. Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative control with TDPs from team TU Delft Protocol: RNase activity Participants: Dawafuti Observations: Results Plate reader experiment with different concentrations of RNA extracted with chemical lysis and phenol chloroform purification from the E.coli culture Protocol: RNase activity Participants: Dawafuti Observations: Results Repeat of plate reader experiment with Lsh Cas13a with Elution 1 and Elution 4,2 Protocol: RNase activity Participants: Dawafuti Observations: Results No activity of Lsh Cas13a visible Cross target plate reader experiment using crRNA E.coli with different target RNAs from B.subtilis and Noro virus Protocol: RNase activity Participants: Dawafuti Observations: Results It didn't work Monday 23rd Plate reader experiment with dried and not dried Cas13a and TDPs with different target concentrations and positive and negative controls Protocol: RNase activity Participants: Dawafuti Observations: Results Plate reader experiment with with Cas13a Lbu and different concentrations of RPA samples Protocol: RNase activity Participants: Dawafuti Observations: Results Plate reader experiment with old Lwa Cas13a elution Protocol: RNase activity Participants: Dawafuti Observations: An elution from July, stored at 4 ºC of Lwa Cas13a was used Results It didn't work Tuesday 24th Plate reader experiment with Lwa Cas13a, old elutions 2 and 3 Protocol: RNase activity Participants: Dawafuti Observations: An elution from July, stored at 4 ºC of Lwa Cas13a was used Results It didn't work Plate reader experiment with cross targets using crRNA form E.coli and different target RNAs form B. subtilis, Noro virus and E.coli Protocol: RNase activity Participants: Dawafuti Observations: Results Plate reader experiment with Spinach aptamer using Lbu Cas13a and different concentrations of in vitro targets Protocol: Participants: Dawafuti Observations: Results It didn't work observation of AuNP linkage asssay with RNA-linker and DNA-linker Protocol: AuNP-DNA conjugation Protocol: AuNP linkage Participants: Rob Observations: To have optimal conditions and high DNA density of DNA on the AuNP for linkage and cleavage, a new AuNP-DNA conjugation was performed. <\li> Over-night linkage was started accoriding to the final protocol. Wednesday 25th Plate reader experiments with lyophilized reaction mix with Cas13a Lbu with different concentrations of the in vitro target. Protocol: Participants: Dawafuti Observations: Cas13a was added only immediately before starting the measurement. Results Plate reader experiment with different concentration of heat lysed E.coli samples with Cas13a Lbu Protocol: Participants: Dawafuti Observations: Results The activity of Cas13a is visible, but additional RNase activity was also seen. General control plate reader experiments with Cas13a Lbu with everything, without crRNA, without target, without Cas13a, positive and negative control. Protocol: Participants: Dawafuti Observations: Results observation of AuNP linkage asssay with RNA-linker and DNA-linker Protocol: AuNP cleavage Participants: Rob Observations: After spinning down the linked AuNP, all supernatant except for 5 μl was removed, pellet resuspended and spotted on paper. <\li> The spotted AuNP were visible as blue aggregates and red, unlinked AuNP. Thursday 26th Plate reader experiment with Noro virus crRNA, Lbu Cas13a and different RNA targets from E.coli, HCV and Noro virus. Protocol: RNase activity Participants: Dawafuti Observations: Results Plate reader experiment with Lwa Cas13a purified from the stored Lwa pellet using different concentrations of invitro transcribed RNAs Protocol: RNase activity Participants: Dawafuti Observations: Results Plate reader experiments with the lyophilized reaction mix on paper using different concentrations of target RNAs Protocol: RNase activity Participants: Dawafuti Observations: Results It didn’t work since the lyophilisation was done overnight which was way too long for a small amount of 15 ul reaction mix Plate reader experiments with the RNA aptamer Spinach with different concentrations of in vitro RNA samples Protocol: Participants: Dawafuti Observations: Results observation of AuNP linkage asssay with RNA-linker and DNA-linker Protocol: AuNP cleavage Participants: Rob Observations: To assure removal of unbound AuNP from , this time, after spinning down the linked AuNP, all supernatant except for was removed, pellet resuspended in 2 μl 1x Cas13a reaction buffer and spotted on paper. <\li> The spotted AuNP were visible as blue aggregates and red, unlinked AuNP. Friday 27th Plate reader experiments with Lwa ( elution 1 and 2 and wash 1 ) purified using Nickel NTA Protocol: RNase activity Participants: Dawafuti Observations: Results Tuesday 31st April May June July August September October