Team:Goettingen/week21-1

V09_20

• 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 of Δtar or BL21 containing pSB1C3 (CM) with a strong promtor and J61002 (AMP) containing a weak promotor (or reverse) were mixed.
• Experimental procedure: View methods.
• Observations and results:
  09_21: Swimming can be observed on all plates with the BL21 constructs! They are scanned. For we found out that our vectord do not contain a ribosomal binding site, the plates containg the BL21 constructs were discarded. Continuation of the experiment with the Δtar strain on 09_22.

• Experiment: In order to produce an overwiew the strains were placed on the different agars
• Experimental procedure: The following plates were poured:
  3% tryptone swimming agar with 100 µl aminoacid mix as attractant (2x)
  M9 swimming agar with 100 µl 0.5% tryptone as attractant (2x)
  The cultures were treated and dropped as described in the methods.
• Observations and results:
  09_21: Swimming of MG and BL21 can be observed on all plates. Plates are scanned.

V09_21

• Experiment:
  In order to produce an overwiew the strains were placed on the different agars
• Experimental procedure: The following plates were poured:
  3% tryptone swimming agar with 100 µl aminoacid mix as attractant (2x)
  M9 swimming agar (+ leucin) with 100 µl 0.5% tryptone as attractant (2x)
  The cultures were treated and dropped as described in the methods.
• Observations and results:
  09_22: On the 3% tryptone swimming agar plates swimming could be expected as expected. The MG strain showed the biggest halo, the one of BL21 was smaller and DH10B and Xl blue did not swimm at all. The plates were scanned. Only very little swimming could be observed on the M9 agar plates. The plates were scanned.

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 (+ leucin) with 100 µl 0.5% tryptone as attractant (2x)
  M9 swimming agar (+ leucin) 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: