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From 2013.igem.org

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Revision as of 13:20, 4 October 2013

Motility assay

A motility assay is done for Bacillus subtilis of different knockout strains as well as a wild type strain.

This is to determine how different the knockouts move compared to the wild type B. subtilis.

The results will be used to determine if the temperature controlled strain works as desired, moving more as a CheY knockout in warm environments and more as a wild type in cold environments.

The design of the motility assay is a simple one. LB agar Plates are made with the normal amount of LB-broth as nutrients but with a reduced amount of agar.

Different concentrations are used to find out the optimal amount of agar to show the motility. Low concentrations are needed to allow movement through the medium but too low and the observed movement can be caused by dispersal and turbulence during movement. The concentrations that are used are, 0.4% agar and 0.7% agar. The plates contained 13 ml agar.

The plates are inoculated in the center with the strain that is to be tested. This is done with 10 ul of liquid culture at an OD₆₀₀ of 0.4. The inoculation was done by sticking the pipette in to the agar, injecting the sample in to it, and by injecting the sample on top of the agar.

For each strain 9 plates of each agar concentration and inoculation method is made. Three of the plates per treatment are placed in a 37°C stove and three placed in a 30°C stove and three placed in a 27°C stove to grow.

The colonies are left to grow for 16 hours. Then how the colonies spread on the plates are observed every hour. The bacteria that are more motile should spread out over the agar creating a cloudy look while the non-motile bacteria should stay in the center, close together.

How quickly the bacteria spread from the center to the edge can be used as an indicator of how fast they move.