Team:Goettingen/week18-1

V08_27

• Experiment:
  The overnight cultures of Δtar_18C_tar and Δtar_18C_rfp were tested on tryptone and M9 swimming agar with methionine as supplement. In addition, the following combinations with antibiotics were tested: with ampicillin (amp), with chloramphenicol (cm), with methionine, with methionine + amp and with methionine + cm. The experiment was performed in three technical replicates.
• Observation & Results:
  The strain Δtar_18C_tar has a resistance against chloramphenicol due to the biobrick plasmid pSB1C3. The strain Δtar_18C_rfp has a resistance against ampicillin due to the plasmid J62100. Both strains did not grow on any of the M9 plates at all. In contrast, both strains displayed growth and swimming on tryptone plates after 1 day. Also chemotaxis was observed. Methione had no influence on swimming or chemotaxis on the tryptone plates.

• Experiment:
  Tested attractants in Whatman paper: caffeine, D-aspartate, geraniol, 2-ethyl-hexanol, vanillin, sodium cyclamate, L-aspartate-4-benzyl. For full description of stocksolutions, look in the materials section. All attractants were used on tryptone and M9 swimming plates with and without chloramphenicol. The library was grown over night. In the morning, the culture was centrifuged 10 min for 1,5k X g. The supernatant was discarded and the pellet was resuspended in the remaining 100 µl LB-medium. 5 µl were dropped on 12 cm petridishes with the different swimming agar. The plates were incubated at 33°C overnight. The experiment was performed in three technical replicates.
• Observation & Results: Swimming was observed for tryptone swimming agar with and without chloramphenicol. M9 plates did not show swimming. Sodium cyclamate, 2-ethyl-hexanol and L-aspartate-4-benzyl showed the highest chemotactic behaviour. Overall chemotaxis was higher on tryptone swimming plates without chloramphenicol.

V08_28

• Experiment:
  Continuation of experiment V_08_27_2. The cultures with the strongest swimming behaviour were cut out at four positions. The agar was recovered for 1 hour in 1 ml LB-medium at 37°C and 180 rpm. Afterwards, the cultures were centrifuged (10 min, 1,5k X g) and the pellet was resuspended in the remaining 100 µl. All strains were plated out on fresh LB-agar plates.

V08_29

• Experiment:
  5 ml LB-medium with the corresponding antibiotics was inoculated with Δtar_18C_tar and Δtar_18C_rfp. A new pellet of the BL21 tar library was used to inoculate 5 ml LB-cm medium. Cultures were grown over night at 37°C and 180 rpm.

V08_30

• Experiment:
  Repetition of V_08_28_1. Afterwards, the resuspended pellets were dropped on new tryptone swimming plates including the different attractants tested in V_08_27_2. This method is described in detail in the methods section.
• Observation & Results:
  Water was used as a negative control attractant. All selected 5 µl strains were dropped on tryptone swimming plates with chloramphenicol. We do not have a reference, yet. An appropiate reference would be E. coli BL21 with the same plasmid pSB1C3 and the same promoter biobrick J23100 (also called 18C for its position on the Distribution plate).

• Experiment:
  The library selection was restarted with a new overnight culture on fresh tryptone swimming plates including the different attractants and water.

• Experiment:
  1,5 ml of overnight cultures were centrifuged for 10 mins at 1.5k X g. The cell pellet was resuspended in the rest media after discarding the supernatant and 5 µl of the strains were dropped on tryptone swimming plates with all aspartate as the attractant. The experiment was conducted in two replicates.

• Experiment:
  tar promoter strength test: Test of the 8 different promoters. Aspartate was used as the attractant in the Whatman Paper. 8 different 5 µl drops were put on the plate on the left side, 3 aspartate Whatman paper were put on the right side in a distance of 3 cm to the drops. The plates were incubated overnight at 33°C.

V08_31

• Experiment:
  The fastest swimming cultures were picked at three positions, recovered for 1 hour in LB-medium and 5 µl were dropped on new tryptone swimming plates including the different attractants and water.

• Experiment:
  The fastest swimming cultures were picked at three positions, recovered for 1 hour in LB-medium and 5 µl were dropped on new tryptone swimming plates including the different attractants and water.

V09_1

• Experiment:
  The first approach of the selction of clones that move directly towards our chosen attractants was selected for a fourth time, bacause no chemotaxis could be observed in the third round.
• Experimental procedure:
  The selection was conducted as described in the methods, but small petridishes (9 cm) were used.
• Observation:
  On the next day swimming could be observed and the plated were treated as described in the protocol "plating of selected clones"

• Experiment:
  One of our goals was to invent es protocol that allows the separation of two different fast strains using the antibiotics marker on an inserted plasmid. The plated prepared at the xx were used to test the separation assay. On these plated cultures of the strain Δtar with J2006 expressing rfp (amp resistance) and the strain Δtar with pSB1C3-tar-QC-18C were mixed 1:1 (according to the OD600) and dropped on 0.3% tryptone swimming agar plates containing no antibiotic.
• Experimental procedure:
  - The agar was cut out at three different positions using a to the first mark shortened yellow Eppendorf tip respectively
  - The first section was cut out at the swimming front (I), and the next two (II, III) in a line behind the first one
  - The tips with the agar pieces were inserted into a glas tube filled with 1 ml LB media, respectively
  - The cultures war incubated for 1 h at 37 °C with approx. 180 rpm
  - a 10^^-1 to 10^^-4 dilution series was prepared
  - 100 µl of the 10^^-2 and the 10^^-4 dilution was plated on LB agar plates containing either ampicillin or chloramphenicol respecvtively
  - the plates were incubated in an 33 °C incubator over night
  
• Observation:
  The next day the colnies could be counted:
  CM containing plates
  
  I: 10^-4: 7 colonies
  II: 10^-4: 320 colonies
  I: 10^-4: 330 colonies
  AMP containing plates
  
  I: 10^-4: 0 colonies
  II: 10^-4: 0 colonies
  I: 10^-4: 7 colonies
  --> the strains could be separated successfully and Δtar with pSB1C3-tar-QC-18C seemed to swimm faster!