Team:Goettingen/week19-1

V09_04

• 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.

• 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

• 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.

• Experiment:
  View methods.
• Observations:
  Continuation in 09_05_1

• Experiment:
  View methods.
• Observations:
  Growth was observed on ever plates, continuation: V09_06_3

V09_05

• Experiment:
  View methods.
• Observations:
  09_06: swimming was observed, experiment was continued

V09_06

• 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.

• 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

• 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

• 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!

• 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!

• Experiment:
  View methods.
• Observations:
  09_08: Growth was observed on every plate, no clones were picked and no vectors were sequenced.

V09_08

• 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.

• 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.