Observations:
Observation: Swimming on every plate, continuation: V09_04_4
V09_03_3: Electron microscopy
Experiment: Experiment: Electron microcopy with the strains BL21 + rfp, +flhDC, +flicCand MG1655.
Observations:
Observation: view focus group 2
V09_04
V09_04_1: Observation of the growth of the strains used for EM, BL21 + rfp, +flhDC, +flicCand MG1655
Experiment: In order to gain a higher cell density which is necessary of EM, the growth of the cells in different incubation conditions was observed. The cells were grown in 5 mL LB-broth.
Observations:
The bacteria grow very slowly and the motility is not that high.
V09_04_2: Quantification of the effect of pSB1C3_tar_QC with different promotors in the strain BL21
Experiment: A standard swimming/chemotaxis assay was conducted with BL21 strains containing tar rescue plasmids with different promotors respectively. View methods.
Observations:
09_05: no swimming was observed
V09_04_3: Separation assay
Experiment: Experiment: The strains Δtar J61002_rfp and Δtar _tar_QC_18C were dropped in the same ratio on 0.3% tryptone swimming agar plates.
Observations:
09_05: only little swimming and no chemotaxis could be observed.
V09_04_4: Second round of selection of the third approach of the tar mutagenesis library
Observations:
09_06: swimming was observed, experiment was continued
V09_06
V09_06_1: Observation of the growth of the strains used for EM, BL21 + rfp, +flhDC, +flicCand MG1655
Experiment: In order to gain a higher cell density which is necessary of EM, the growth of the cells in different incubation conditions was observed. The cells were grown in 5 mL LB-broth, in 5 mL LB agar topped with 5 mL LB broth, in 10 mL LB-broth and in 10 mL LB agar topped with 10 mL BL-broth.
Observations:
After 5.5 h an OD600 of over 2 in 10 mL LB agar topped with 10 mL BL-broth can be reached but it is suspected, that the expression of flagella is reduced through the shaking in the erlenmyer flask. Light microscopic test have to follow.
V09_06_2: Separation assay
Experiment: The strains Δtar J61002_rfp and Δtar _tar_QC_18C were dropped in the same ratio on 0.3% tryptone swimming agar plates.
Observations:
Continuation: 09_08_2
V09_06_3: Selection of clones from the “plating on the chloramphenicol containing LB plates step” for sequencing and observation of the swimming behavior
Experiment: Clones were selected form the CM plates containing clones form the library. These were again secured on LB agar plates (with CM) and used to inoculate 7 mL LB-broth containing CM..
Observations:
09_07: all cultures grew, the minipreparation was conducted and the extracted vectors transformed into fresh BL21 cells.
V09_07
V09_07_1: Observation of the swimming behavior of the through the library selection selected clones after they were plates on LB-agar containing chloramphenicol
Experiment: In order to investigate whether the selected clones containing a library vector still swim with approximately the same speed after they have been plated an LB agar containing chloramphenicol they were dropped again on 0.3 % tryptone swimming agar with the respective attractant and incubated over night at 33 °C
Observations:
09_08: The clones still swim as fast!
V09_07_2: Retransformation of the extracted plasmids of the selected clones containing the library vectors into fresh BL21 cells
Experiment: In order to determine whether the observed behavior is dependent on the selected clones or on their vectors, the extracted vectors were transformed into new BL21 cells. View methods.
Observations:
09_08: growth was observed on each plate!
V09_07_3: Plating of the selected clones of the 3rd approach of the library selection
Observations:
09_08: Growth was observed on every plate, no clones were picked and no vectors were sequenced.
V09_08
V09_08_1: Swimming behavior of the library: selected clones versus retransformed clones
Experiment: Continuation of 09_07_1. Growth could be observed on every plate, single colonies were picked and used to inoculate 1 ml LB broth. Cultures were shaken for approximately 3 h.
Observations:
09_09: there is a huge difference between the swimming behavior of the selected clones and the retransformed clones.
V09_08_2: Separation assay
Experiment: Separation of the strains Δtar J61002_rfp and Δtar _tar_QC_18C. The agar was cut out at three different positions and the pieces incubated in 1 mL LB broth for approximately 3 h. The cultures were diluted and the 10^-2 and the 10^-4 dilution was plated on plates containing either ampicillin of chloramphenicol. 3 dopes were treated as described above.
Observations:
09_09: In one of three cases (one not evaluable) the Δtar _tar_QC_18C strain was faster.
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