Team:Goettingen/week13-1

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#1 Selection / Swimming - 13th Week

Fluorescence diffusion test

Experiment:
Tryptone solution was supplemented with fluorescin to identify the optimal fluorescin concentration for the visualization of diffusion of the chemoattractant soaked into the whatman paper. Therefore, plates with M9 agar were prepared, the whatman paper in the middle of each plate was soaked with tryptone and in addition a variety of amounts of fluorescin. All that was done for several incubation temperatures (RT, 4°C, and 37°C)
Observations & Results:
After two hours, no diffusion was visible on any plate at all.

Separation approach

Experiment:
Equimolar mixtures of a great variety of strains with a lot of different plasmids were dropped onto M9- and tryptone-agar plates. After chemotaxis is visible on the plates, the bacteria from the most outer front will be picked and resuspended. This resuspension will be streaked out onto new plates and counted, to determine the ratio of the mixed strains. This information can be used, to identify the faster and more motile strains.
Observations & Results:
After two days, the plates showed no significant swimming. After one more day, swimming was detectable for BL21 and some DH10B strains. Only BL21 showed notable chemotaxis, whereas the kind of transformed plasmid doesn't seam to have any major influence. All BL21 straines showed more or less the same amount of chemotaxis.

Repetition of the fluorescin diffusion assay (V07_23)

Experiment:
V07_25: A variety of dilutions (1:5, 1:20, 1:50, and 1:100) of the fluorescin stock solution with aspartate solution were produced. These solutions were incorporated into the whatman paper and plated onto the M9 agar plates. Now, a picture was taken from time to time to identify both, the perfect concentration and duration for diffusion. Everything was incubated at 37°C
Observations & Results:
After 20h, the dilution levels 1:20 and 1:50 showed good results. Not everything, but more then the half of the plates were taken by diffusion.

Preparation of glycerol stocks of transformed cells

Experiment:
Strains of BL21 and DH10B with a variety of plasmids (mostly different promotor strengths and Tar or flhDC ORF downstream) were converted into glycerol stocks and stored at -80°C.