Part:BBa_K343004:Experience

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Applications of BBa_K343004

Motility assay

Exp. 1

The purpose of this experiment is to see if the cells containing pSB1C3-K343004 move farther than the wild type (MG1655) and the negative control (DH5alpha). This would be an indication of hyperflagellation.We also want to test if it makes a difference in the motility wether the bacteria contain low-medium- or high-copy plasmids.

For the motility experiments we added 5ul of an ON culture to petridishes containing motility agar (LB media with 0.3% agar) instead of regular LA (Luria agar). This semifluid media lets the bacteria swimm more easily.
The plates were left in an incubator (37 degrees) for 24 hours.





The upper two plates did not contain antibiotics, and therefore contamination colonies are seen.
The upper left picture is of E. coli strain DH5alpha that does not express flagella and therefor movement in the media should, as seen on the picture, be minimal. The upper right picture is of the wild type E. coli strain MG1655, this strain has about 8-10 flagella per cell. These cells are, as seen, expected to move farther than the DH5alpha but not as far as the transformed cells. The lower left picture is of E. Coli strain MG1655 with pSB1C3-K343004 this shows that these bacteria move farther than the wild type and the negative control. The lover right picture is of E. coli strain MG1655 with pSB3K3-K343004 these bacteria move farther than the wild type, the negative control and MG1655 with pSB3K3-K343004.

From the pictures above we can definately se that the bacteria containing our part is much more motile than the wild type. We assume this is caused by overexpression of the FlhDC master flagella operon which leads to hyperflagellation of the cells.
The two buttom pictures show that bacteria with pSB1C3-K343004 have not moved as far as the bacteria containing pSB3K3-K343004. pSB1C3 is a high copy plasmid while pSB3K3 is a low-medium copy plasmid. The promoters in K343004 is a constitutive promoter (tetR repressable promoter). Bacteria containing a high copy plasmid with a constitutive promoter are more metabolically challanged than bacteria containing a low- or medium-copy plasmid with a constitutive promoter because of the higher number of plasmids per the cell. Therefore the high copy plasmid bacteria are less motile than low- or medium-copy plasmid bacteria.

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