Team:Goettingen/week14-2

V07_30

• Experiment:
  In order to perform a further swimming assay new over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in DH10B, BL21, DH5alpha and XL1 Blue were prepared.
  

V08_01

• Experiment:
  In order to additionally consider the two new motile E. coli strains MG1655 and RP437 in the swimming assay new over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in DH10B, BL21, DH5alpha and XL1 Blue and of MG1655 and RP437 were prepared.
  

V07_10

• Experiment:
  LB medium containing ampicillin was inoculated with the over night culture and shaken until an optical density of 0.6-0.8 was reached. Meanwhile ampicillin containing M9 agar plates were prepared and autoclaved Whatman Filter Papers were imbued with tryptone solution. Then the cultures were dropped on the agar at a distance of 1 cm to the paper. On each plate also colonies hosting the empty vector pUC18 were dropped in order to have a reference. Two drops were rather applied into the agar than onto as done before, because some paper predicted faster motility. Also this time each plate was prepared in duplicates.
  
• Observations & Results:
  Here again a certain diversity in the growth rate could be observed. Whereas some cultures had already reached and optical density of 0.8, others were still at merely 0.04.Here again fliC featured a low density, this time in all strains and DH10B and BL21 show a general decreased growth rate. Striking is the fact that the strains that featured the best motility as well as the construct that seems to have the biggest influence show decreased division rate. Whether this phenomenon is a coincidence or not we are not sure about yet. At the following day (26th of July) as well as the day after (27th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed. Almost a weak later (30th of July) still no swimming or chemotaxis had taken place, solely growth. However, the two colonies that had been applied into the agar not onto featured little swimming. Nevertheless, also these results are lousy considering the long time span. (

V07_11

• Experiment:
  For the chemical transformation the standard protocol was followed.
  
• Observations & Results:
  The transformation was successful since all plates showed numeours colonoes except the negative control.

V07_12

• Experiment:
  Over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in pUC18 were prepared in order to isolate the plasmids.
  

V07_13

• Experiment:
  Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

• Experiment:
  All vectors were digested with EcoRI and XbaI according to the protocol. The restriction products were subsequently separated on a gel in order to determine successfully ligated plasmids.
  
• Observations & Results:
  Except for fliC (Salmonella) #1 all picked colonies seem to host plasmids containing the aimed insert.