Team:Goettingen/week21-1

V09_17

• Experiment:
  For the Separation assay from the 09_16 was not conducted according to the protocol it was repeated.
• Experimental procedure: The cultures were treated as described in the methods.
• Observations and results:
  09_18: The drops did not turn red thus we assumed, that the plasmid was discarded. The plates were dumped.
  

• Experiment:
  To observe the chemotaxis of the Δtar with pSB1C3_tar_QC_18C a capillar assay was conducted.
• Experimental procedure:
  100 µl of the culture (OD600=0.45) was mixed with 1400 µl chemotaxis buffer and placed on a microscopic slide. Capillarys containing either aspartate or chemotaxis buffer were placed on each side of the drop. The approach was covered with a cover spil and fixed with nail polish. After 50 minutes the capillarys were broken and put into an e-cup containing 200 µl LB-broth. The e-cups were centrifuged for 1 min at 5000 x g and the dilutions 10_-1 to 10_-3 were plated on LG agar plates containing chloramphenicol. .
• Observations and results:
  09_18: More colonies could be counted on the LB plate which contained the cells from capillary with aspartate than on the plates which contained the cells from the capillary with chemotaxis buffer. Because the experiment was only conducted once it was repeated on the 09_18.

V09_18

• Experiment:
  In order to be able to directly compare the "trafos" and "retrafos" the clones were dropped on plates containing the attractend as well as on 2 additional control plates.
• Experimental procedure: The overnight cultures of the clones ( view methods for nomenclature) were treated as described in the described in the methods. Each clone was dropped on a plate containing the attractant, H20 and L- aspartate (controls) respectively.
• Observations and results:
  09_19: Swimming was observed on every plate. In general the "trafos" showd a better swimming behaviour than the "retrafos". In most cases even the reference strain swam faster than the "retrafos". All plates were masured. We suspect that through our selection system we selected strains containing a certain receptor as well as in general fast strains. The next step should be selection of the "retrofos" after three rounds of selection the "retrafos" should be as fast as the "trafos".

• Experiment:
  To observe the chemotaxis of the Δtar with pSB1C3_tar_QC_18C a capillary assay was conducted.
• Experimental procedure:
  100 µl of the culture (OD600=0.45) was mixed with 1400 µl chemotaxis buffer and placed on a microscopic slide. Capillarys containing either aspartate or chemotaxis buffer were placed on each side of the drop. The approach was covered with a cover spil and fixed with nail polish. After 50 minutes the capillarys were broken and put into an e-cup containing 200 µl LB-broth. The e-cups were centrifuged for 1 min at 5000 x g and the dilutions 10_-1 to 10_-3 were plated on LG agar plates containing chloramphenicol. .
• Observations and results:
  09_19: Growth was observed on each plate. In 2 of three cases the approach containing aspartate showed a higher number of colnies.

• Experiment:
  For in the approach of the 17.09 the drops did not turn red (probably the vector was lost) the eperiment was repeated.
• Experimental procedure: Different amounts of aspartate were applied to the whatmanpaper: 10 µl, 25 µl, 50 µl, 75 µl and 100 µl. View methods.
• Observations and results:
  09_19: No swimming could be observed.
  09_20: Still no could be observed, we assume that to much agar was added and thus the agar was too solid to enable swimming.

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.

• Experiment: IN order to produce resultes that can be used in a figure the separation assay was repeated.
• Experimental procedure: THe cultures were applied to 0.3% tryptone swimming agar as well as M9 agar. Two different amouts of atpartate were used (50 µl and 100µl) ao overall 8 plates were poured. View methods. .
• Observations and results:
  Continuation in V09_22_5.

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 containend methionine and were already a day old before use, aspartate was used as attractant.
• Observations and results:
  09_23: Swimming could be observed for the BL21 strain but not for the Δtar strain. THe swimming of the BL21 strain does not seem to be directed. 09_24: Swimming can be observed for the Δtar strain. It seems to be directed and promotor dependent.

• 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:
  23_09: the sreins did not swim as expected and thus this experiment was not completed.

• 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: