Team:Goettingen/week12-2


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#2 Speed Improvement - 12th week

Chemical retransformation of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH into E. coli (DH10B, BL21, DH5α and XL1 Blue)

Experiment:
For the chemical retransformation the standard protocol was followed. This time the four E. coli strains DH10B, BL21, DH5α and XL1 Blue were transformed in order to subsequently perform swimming assays and compare the accessibility of the different strains.
Observations & Results:
The retransformation was succesful. Numerous colonies had formed at each plate except for the negative control.

Preparation of over night cultures

Experiment:
In order to test the influence of the different genes on the motility of the distinct strains over night cultures were prepared. On that account it is important to shake the cultures only at 140 rpm to spare the flagella.
Observations & Results:
Not all cultures grew properly. Especially, those of E.coli BL21 featured a low cell density.

Performance of motility assay

Experiment:
LB medium containing ampicillin was inoculated with the over night culture and shaken until an optical density of 0.6 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. Each plate was prepared in duplicates.
Observations & Results:
The following freshly inoculated cultures did not grow very well and only reached an OD between 0.3 and 0.4:
fliC (DH10B) in DH10B
fliC (DH10B) in DH5α
fliC (DH10B) in XL1 Blue
motB in DH5α
motB in XL1 Blue

At the following day (19th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed. Two days after preparation of the swimming assay plates (20th of July) slight chemotaxis and motility could be perceived. motB and fliC overexpression seems to have a positive influence on bacterial motility in all strains. At the 23rd of July quite big halos had formed. motB shows a strong effect, especially in DH10B. fliC (DH10B) seems to increase motility in all strains. However, the difference to the reference is not very drastic at all, but since the genes are not under control of strong promoters this results seems reasonable.