Team:Goettingen/week20-1

09_14

• Experiment:
  The library was applied to the 0.3% tryptone swimming agar for selection
  View methods.
• Observations:
  09_16: Because of group internal misscommunication the library was not further selected but applied to LB-plates containing chloramphenicol. Continuation: V09_16_1.

• 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 plates prepared at the V09_14_2 were used to test the separation assay. On these plates 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 anibiotic.
  The cultures were mixed and applied to 0.3% tryptone swimming agar containing different amounts of aspartate respectively (2 x 10 µl, 2 x 25 µl, 2 x 100 µl). The mix was dropped on the plates two times and Δtar with pSB1C3-tar-QC-18 and Δtar with J2006 expressing rfp once each.
• Observations:
  Continuation: V09_16_1

• Experiment:
  We suspected that the rfp expression might influence the cell density measurement and to invest this the following experiment was conducted.
  The strains Δtar with J2006 expressing rfp and Δtar with pSB1C3-tar-QC-18C were inoculated in the morning and diluted (10^-1, 10^-2, 10^-3, 10^-4, 10^-5) in the afternoon. The OD600 was measured and the cultures subsequently plated on LB agar plates.
• Observations:
  In general the number of colonies of the rfp expressing strain is always much lower than in the other strain even though the measured OD600 is much higher! The rfp expression influences the OD600 measurement!!!

• Experiment:
  It was ssuspected, that the amount of aspartate influences the swimming behaviour of the tar rescue strain
  The strains Δtar with J2006 expressing rfp and Δtar with pSB1C3-tar-QC-18C were dropped on plates containing 0.3 % tryptone swimming agar and whatmanpaper soaked with different volumes of the aspartate solution (10 µl, 25 µl, 100 µl)
  View methods.
• Observations:
  09_15: no different tendencies could be observed.

• Experiment:
  It was ssuspected, that the amount of agar in a petrish influences the swimming bahviour in general.
  Δtar with pSB1C3-tar-QC-18C was dropped on plates containing different amounts of 0.3% tryptone swimming agar (15 ml, 20 ml, 25 ml, 40 ml). Small9 cm petridishes were used.
  
• Observations:
  09_15: no different tendencies could be observed.

09_16

• Experiment:
  Because of group internal misscommunication the library was not further selected but applied to LB-plates containing chloramphenicol
  
  View methods.
• Observations:
  09_17: Growth was observed on every plate. They were placed in the fridge. No clones were picked for sequencing.

• Experiment:
  In order to qualtify the swimming behaviour the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors are used for a standard swimming/cemotaxis assay
  100 µl Aspartate solution on the whatmanpaper was used as an attractant and 0.3% tryptone swimming agar was used.
  View methods.
• Observations:
  09_17: No swimming could be observed.

• Experiment:
  In order to qualtify the swimming behaviour of the unmodified strains they are placed on swimming agar
  The strains were placed on 0.3% tryptone swimming agar and M9 agar containing either aspartate, the aminoacid mix or the tryptone solution in the whatmanpaper (concentrations: view materials. The standard swimming/chemotaxis assay was applied, view methods.
  
• Observations:
  09_17: No swimming could be observed.

• 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 plates prepared at the V09_14_2 were used to test the separation assay. On these plates 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 anibiotic.
  - The agar was cut out at the swimming front using a to the first mark shortened yellow Eppendorf tip respectively. Two different mixed cultures and one containing pSB1C3-tar-QC-18C or J2006 expressing rfp only.
  - The tips with the agar pieces were inserted into an e-cup filled with 1 ml LB media respectively
  - The cultures were incubated for 1 h at 37 °C with approx. 180 rpm
  - A 10^-1 to 10^-4 ditutions 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 cultures containing pSB1C3-tar-QC-18C or J2006 expressing rfp only were only plated on cm or amp plates.
  - the plates were incubated in an 33 °C incubator over night.
  
• Observations:
  - Mix #1:
  
  Cm: 10_-2: 1284
  Cm: 10_-4: -
  Amp: 10_-2: 1460
  Amp: 10_-4: -
  - Mix 2#:
  
  Cm: 10_-2: 14
  Cm: 10_-4: 1
  Amp: 10_-2: -
  Amp: 10_-4: -
  - pSB1C3-tar-QC-18C only
  
  Cm: 10_-4: 76
  - J2006 expressing rfp only
  
  Cm: 10_-4: 112