Motility test

Motility assay

To test the difference in motility between the wild type Bacillus subtilis 168 strain and the both knockout strains, ΔcheY and ΔcheYΔdes, a motility test is done.
The plates are inoculated in 25°C and 37°C to determine the different behaviour of the strain at different temperatures, as these are the temperatures we expect changes when the Pdes-cheY construct is inserted into the ΔcheYΔdes strain. In general we expect the wildtype strain to be more motile than the knock out strains. The design we used for our motility assay is a simple one. The LB agar plates are made with the normal amount of LB-broth as nutrient, but with a reduced amount of agar. Low concentrations of agar are needed to allow movement through the medium, but when the concentrations are getting too low the observed movement can be caused by dispersal and turbulence during movement.
So we decided to use LB agar plates with a concentration of 0.4% of agar. For the reproducibility of the project, it is decided to pipet 13 ml of agar to all of the plates. On every plate 10 µl of liquid culture with an OD₆₀₀ of 0.4 is pipetted.
The assay is performed in triplo for each strain and the plates are inoculated for 16 hours. The bacteria that are motile should spread out over the agar creating a cloudy look while the non-motile bacteria should stay at the spot. How quickly the bacteria spread from their spot to the edge can be used as an indicator of how fast they move.

Microscopy

Pictures were taken with a phase-contrast microscope every second for 2 min with an exposure to white-light of 0,025 seconds.

To test the cheY and des knockout

For our heat motility model we need at least a double knockout strain of B.subtilis. A knockout of both cheY and des are necessary. To obtain the double knockout strain, first a knockout of cheY is made. This is done by transforming the complete genomic DNA of the cheY mutant of Ordal to the 168 strain [1]. After obtaining this mutant strain, des knockout is inserted.

Correct insertion of the des knockout

A knockout of gene des is inserted into the genomic DNA of B.subtilis strain 168 with a tetracyclin resistance marker. Colony PCR showed that des is indeed transformed into the genomic DNA (Figure 1).

Figure 1: Colony PCR of the des knock out

Motility of the knockout strains

To observe whether or not the mutant strains are less motile than the wild type strain. Two different tests are done.

Motility assay

To compare the motility of the wildtype strain with the two knockout strains, ΔcheY and ΔcheYΔdes, a motility assay is made. All the results obtained from this assay is from an experiment performed in triplo. When the strains are grown on a 0.4% LB agar plate, after 16 hours of growth it is visible that the wildtype strain shows more swimming behaviour than both of the mutant strains (Figure 2). A comparison between the two mutant strains indicates that ΔcheY has better swimming behaviour than ΔcheYΔdes.

Figure 2: Motility assay results after 16 hours of growth

When the plates are incubated for 21 hours, it is more distinguishable that the wildtype strain shows more swimming behaviour than the mutant strains. A comparison at two temperatures, 25°C and 37°C, is made. A comparison between the strains at both temperatures shows that the wildtype strain swims better than the ΔcheY strain and the ΔcheY swims better than the ΔcheYΔdes strain. It is also visible that cells grow better when they are in a warmer environment (Figure 3).

Figure 3: Motility assay results after 16 hours of growth at 25°C and 37°C.

Microscope movies

Another way of analyzing the swimming behaviour is to make microscope movies (4x real time). These movies are made for the wildtype, the ΔcheY and the ΔcheYΔdes strain. These movies show that the wildtype strain (Movie 1) is more motile than both mutant strains. The comparison between the movie of the ΔcheY (Movie 2) and ΔcheYΔdes (Movie 3) strain shows just as seen in the motility assay, that the ΔcheYΔdes strain is less motile than the ΔcheY strain.

Movie 1: Motility of the wild type strain. A constant flow of cells is visible with cells swimming against/through the stream, indicating for motile bacteria.

Movie 2: Motility of ΔcheY. A constant flow of cells is visible with one cell tumbling around his own axis on a fixed position. Indicating a non-motile cell.

Movie 3: Motility of ΔcheYΔdes. Cells seem to be non-motile during this time-lapse.

[1] Rosario, M. M., et al. "Chemotaxis in Bacillus subtilis requires either of two functionally redundant CheW homologs." Journal of bacteriology 176.9 (1994): 2736-2739.