27.7.2022: meeting with MedChemExpress, Drylab: meeting with Prof. Thiel
28.7.2022: Wetlab: propagation of competent NEB5alpha cells for interlab study
29.7.2022: Wetlab: redo propagation of competent NEB5alpha cells for interlab study
30.7.2022: video: First meeting, discussion of equipment, roles, schedule
31.7.2022: Impact grand awarded, Human Practices meeting
Week 31▼
1.8.2022: weekly team meeting, Wetlab: interlab study: culture NEB5alpha cells and calibrate plate reader; PctD-LBD expression: SDS gel to check expression; start generating competent VS994 cells
2.8.2022: Wetlab: interlab study: continue preparation of competent cells, prepare buffers for purification
3.8.2022: Wetlab: agarose gel for colony PCRs
4.8.2022: Wetlab: interlab study: check transformation efficiency of competent cells; purification of D-PctD seems successful
5.8.2022: Wetlab: transformation of competent cells failed
6.8.2022: video: Meeting with Dr. Khaled Salim to discussion of venues for shooting and shooting in the lab
Week 32▼
8.8.2022: weekly team meeting
9.8.2022: Wetlab: SDS page of PctD-LBD purification, agarose gel of PCRs, interlab study: transformation with interlab constructs
10.8.2022: Wetlab: interlab study: transformation failed, redid with purchased NEB5alpha competent cells, video: Meeting of the whole team to take team drone shots
11.8.2022: Wetlab: interlab study: transformation of constructs 1,2,3 successful -> continued incubation; preparation of competent cells continued, plasmid isolation of VS1001, preculture of VS1007 failed
12.8.2022: Wetlab: meassure concentration of purified PctD-LBD -> total yield of app. 1.75mg protein, interlab study: after 40h of incubation colonies for all constructs are present, Human Practices meeting
14.8.2022: Human Practices meeting
Week 33▼
15.8.2022: weekly team meeting, Wetlab: calibration for interlab study, video: Dicussion of differents variants of text for the script; discussion which shots to use for which part of text, discussion of animation for parts of our video
16.8.2022: Wiki/Design meeting, wetlab: interlab study. Redo transformations with constructs; creation of chimeric receptor: transformation of NEB5alpha with pKG116 plasmid
17.8.2022: Wetlab: interlab study: “old” colonies fluoresce under microscope -> use for further experiment, new plates have no colonies -> incubate further; Creation of chimeric receptor: grow cells for plasmid prep of pKG116; gradient PCR of EnvZ and EnvZ-HAMP shows corect amplification of EnvZ-HAMP but not EnvZ
18.8.2022: Wetlab: interlab study: meassurment of constructs; PCR of PctD, PctD-HAMP and pKG116 shows correct amplification of PctD and PctD-HAMP but not pKG116
19.8.2022: Wetlab: interlab: repeat meassurment of constructs; gradient PCR of EnvZ and pKG116
Week 34▼
22.8.2022: weekly team meeting, Launch overhauled website, Wetlab: preculture for new PctD-LBD expression, gradient PCR of EnvZ failed, video: Filming of cyanobacteria in culture and under the microscope in the laboratory; final script discussion (camera shots, text, ...)
23.8.2022: Gibson Assembly Kit bought, Wetlab: main culture and harvesting of cells for PctD-LBD purification, EnvZ gradient PCR with Q5 polymerase works, video: Shot sequence of talking person in the lab (Part "Deep project dive")
24.8.2022: Drylab: Meeting with team from Greece, visiting the central public health authority of Baden-Württemberg
25.8.2022: Wetlab: purification of PctD-LBD shows 2 peaks that could contain PctD-LBD
26.8.2022: video: Final script corrections, choosing of background speaker, discussion of questions regarding animation, editing, cutting, subtitles, attributions
Week 35▼
29.8.2022: weekly team meeting, Wetlab: SDS page of PctD-LBD purification -> protein contained in one peak, fraction pure, dyalisis of protein fractions against ITC buffer, chimeric chemoreceptor generation: purification of PCR products and Gibson Assembly, transformation of assembly product
30.8.2022: Wetlab: PctD purification: meassure protein concentration, chimeric chemoreceptor generation: transferred 20 colonies per construct (1 and 2) on new plates
31.8.2022: Wetlab: chemoreceptor generation: colony PCR of 20 colonies per construct
Video: Final discussion about editing, subtitles, attributions
1.9.2022: Wetlab: chemosensor generation: agarose gel of colony PCR shows positive control for most colonies of contruct 2 -> overnight culture of positive colonies, meeting with Libera
2.9.2022: Wetlab: chemoreceptor generation: plasmid isolation of selected PCR-positive colonies, video: Uploaded final version
Week 36▼
5.9.2022: weekly team meeting
6.9.2022: Wetlab: sent plasmids to sequencing, started propagation of cells (VS996, VS1001) for chemically competent E.coli, Drylab: meeting with team Patras
7.9.2022: Wetlab: continuation of preparation of chemically competent E.coli
8.9.2022: Wetlab: continuation of preparation of chemically competent E.coli, test transformations with pUC19, received sequencing results, Human Practices survey in Tuebingen
Week 37▼
12.9.2022: weekly team meeting, Wetlab: continuation of transformation of VS996 and VS1001 with pUC19 and VS1008 and pUC19 and PctD-HAMP-EnvZ respectively
13.9.2022: Wetlab: checking results of transformations, plasmid isolation of VS1001 and sending for sequencing, Gibson assembly of PctD-EnvZ-Hamp and PctD-Hamp-EnvZ and transformation into NEB5alpha competent cells
14.9.2022: Wetlab: preparation of chemically competent VS1007 cells, colony PCR and re-streaking of transformed cells from the day before
15.9.2022: Wetlab: gel electrophoresis of colony PCR, preparation of Choline chloride stock for ITC measurement
16.9.2022: Wetlab: ITC meassurement of PctD-LBD, plasmid isolation of transformed cells and sending for sequencing
Week 38▼
19.9.2022: Wetlab: received sequencing results, transformation of the hybrid receptor constructs (PctD-EnvZ-Hamp and PctD-Hamp-EnvZ)
28.9.2022: Wetlab: SDS Page of VS1001 PEH, VS1001 PHE and VS1007 PEH, Wiki: Planning meeting and first implementations
29.9.2022: Wetlab: test of GFP expression of the biosensor: fluorescence measurement of VS1001, VS1001+PEH, VS1001+PHE, VS1007, VS1007+PEH, VS1007+PHE after induction with Aspirin, Human practices: Visit to the chemical testing facility CVUA Sigmaringen
30.9.2022: Wetlab: repeated test of GFP expression of the biosensor, Wiki: implementation session
2.10.2022: Wiki: discuss further To-Dos
Week 40▼
3.10.2022: weekly team meeting
4.10.2022: Wetlab: resistance testing of VS1007, preparation of glycerol stock of VS1007-PEH
5.10.2022: Wetlab: checking resistance test of VS1007, continuation of preparation of glycerol stock of VS1007-PEH, preparation of stock solutions for acetylcholine, choline, acetylsalicylic acid, prepared quantitative testing of biosensor VS1007-PEH
6.10.2022: Wetlab: continuation of testing of biosensor VS1007-PEH failed (no bacterial growth), troubleshooting for testing of biosensor, prepare new testing of biosensor
7.10.2022: Wetlab: quantitative testing of biosensor VS1007-PEH with plate reader over period of 4h