Team:Goettingen/week17-1

V08_20

• Experiment:
  Group two constructs (FlhDC, FliC (DH10B), FliC (Salmonella), MotA, MotB, yhjH) have been cloned into the biobrick vector pSB1C3 and were chemically transformed in compentent DH10B and BL21 following the standard protocol. The empty vector pSB1C3 was transformed, too.

• Experiment:
  MG1655 strains from plates of V_08_17_1 were cut out 2 times. The agar was incubated in 1 ml LB-medium at 37°C and 180 rpm for 1 hour. The cultures were spun down for 10 mins at 1,5k X g. The supernatant was discarded and the pellet was resuspended in the remaining LB-medium. Ca. 7 µl were dropped on fresh trypton swimming and M9 plates with AS-mix in the Whatman Paper. Subsequently, the plates were incubated at 35°C.

V08_21

• Experiment:
  Same procedure as in V_08_20_2. The selected cells were dropped on tryptone swimming plates with AS-mix in the centeral Whatman paper.
• Observations & Results:
  Selection was finished after this round. The fast strains were picked and plated on LB-plates and stored at 4°C.

V08_22

• Experiment:
  5 ml LB-medium with ampicillin was inoculated with Δtar_18C_tar and Δtar_18C_rfp. 5 ml LB-medium was inoculated with E. coli DH10B and BL21 containing various group 2 constructs with the corresponding antibiotics.

V08_23

• Experiment:
  The separation assay was conducted in different approaches:
  1. Overnight cultures of the strains Δtar_18C_tar and Δtar_18C_rfp were used in the morning to inoculate 5 ml LB-medium with ampicillin and shaken at 37°C and 180 rpm. After 7 hours of growth the cultures were a) dropped out directly and b) 1.5 ml of the cultures were centrifuged for 5 mins at 5 X g.
  2. The overnight cultures were a) dropped out directly and b) 1.5 ml of the cultures were centrifuged for 5 mins at 5 X g. Both cultures were mixed equally according to the OD600 measurement. 5 µl of two times the mixture and both cultures independently were dropped on fresh M9 plates with the attractant aspartate for each attempt. The experiment was conducted in two replicates.
• Observations & Results:
  No swimming was visible. Most cultures displayed only minor growth. Problems with temperature or over night cultures assumed.

• Experiment:
  The motility assay was conducted in different approaches:
  1. DH10B and BL21 overnight cultures were used in the morning to inoculate 5 ml LB-medium with ampicillin and were shaken at 37°C and 180 rpm. After 7 hours of growth the cultures were a) dropped out directly and b) 1.5 ml of the cultures were centrifuged for 5 mins at 5 X g.
  2. The overnight cultures were a) dropped out directly and b) 1.5 ml of the cultures were centrifuged for 5 mins at 5 X g. 5 µl of each strain were dropped on fresh M9 plates with the attractant aspartate for each attempt. The experiment was conducted in two replicates.
• Observations & Results:
  Most cultures displayed only minor growth. If swimming was observed, than only for BL21 strains. Constructs FlhDC, FliC (DH10B) and MotB in BL21 showed swimming behaviour. The swimming had no obvious chemotaxis.

V08_24

• Experiment:
  Repetition of V_08_23_1.

• Experiment:
  Repetition of V_08_23_2.
• Observations & Results:
  BL21 strains showed stronger swimming than DH10B. Especially, the constructs FlhDC, yhjH and MotB in BL21 showed swimming behaviour. Chemotactic behaviour was observed in very few cases.

V08_26

• Experiment:
  E. coli DH10B and BL21 with various group two constructs were used to inoculate 5 ml LB with the corresponding antibiotics. 5 ml LB-medium with the corresponding antibiotics was inoculated with Δtar_18C_tar and Δtar_18C_rfp. Cultures were grown over night at 37°C and 180 rpm.