Team:Goettingen/week21-1

V09_22

• Experiment:
  In order to produce an overwiew the strains were placed on the different agars with and without an attractant
• Experimental procedure: The colonies were inoculated in the morning and grown during the day for nearly 6 hours. Each strain was plached on each plate and the following plates were poured
  3% tryptone swimming agar with 100 µl aminoacid mix as attractant (2x)
  3% tryptone swimming agar without attractant (2x)
  M9 swimming agar with 100 µl 0.5% tryptone as attractant (2x)
  M9 swimming agar without attractant (2x)
  The cultures were treated and dropped as described in the methods.
• Observations and results:

• Experiment: The tar fragment in in the vectors pSB1C3 (CM) and J61002 (AMP) with different promotors. In order to test the effect of the promotor strength cultures containing pSB1C3 (CM) with a strong promtor and J61002 (AMP) containing a weak promotor (or reverse) were mixed. This procedure was executed with the Δtar and the BL21 strain, but for no ribosome binding site was added, the probability to gain a result was higher with the Δtar strain, for this one is tar deficient. The cultures showed chemotaxis befor cutting of the agar.
• Experimental procedure: view methods.
• Observations and results:

• Experiment: In order to qualtify the swimming behaviour and to find ouf whether the promotors have an effect even without ribosomal binding site the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors are used for a standard swimming/cemotaxis assay .
• Experimental procedure: view methods. The plates were already a day old before use, aspartate was used as attractant.
• Observations and results:

• Experiment: In order to control the separation assay the setup was repeated with strains whose swimming behaviour is not dependent on the expression of a vector for al our vectors are lacking a ribosomal binding site. Here the strain MG containing pRSB1C3_18C_rfp (CM) and BL21 containing J61002_18C_flhDC (AMP, useless insert)were used.
• Experimental procedure: view methods. The drops were applied on the following plates:
  3% tryptone swimming agar with 100 µl aminoacid mix as attractant (2x)
  3% tryptone swimming agar without attractant (2x)
  M9 swimming agar with 100 µl 0.5% tryptone as attractant (2x)
  M9 swimming agar without attractant (2x)
• Observations and results:

• Experiment: Even though no ribosomal binding site is present on can assume that at least a few or the tar mRNAs are translated and thus the TAR-receptor is restored. To confirm the separation assay it was tested again.
• Experimental procedure: The mixed cultures as well as the Tar rescue strain exibited chemotaxis whereas the Δtar strain ecpressing rfp did not. View methods.
• Observations and results: