This article describes a method for separating bacterial strains based on their capsule amounts using a discontinuous density gradient. The technique allows for the analysis of bacterial transposon mutant libraries, facilitating downstream studies of bacterial genetics.
Begin with a tube containing a pre-set discontinuous density gradient, prepared using non-toxic colloidal silica particles coated with polyvinylpyrrolidone.
The gradient comprises three non-overlapping layers arranged in order of increasing densities from top to bottom.
Take a bacterial transposon mutant library, a pooled suspension of bacterial strains with random transposon-induced mutations.
A subset of these mutations disrupts genes that regulate the capsule, a polysaccharide layer surrounding the bacterial cell wall, resulting in strains with varying capsule amounts.
Add the bacterial library to the top of the gradient very slowly, without mixing the interface.
Centrifuge the tube at a moderate speed.
The gradient facilitates the differential migration of bacteria based on their capsule amounts.
Hyper-capsulated strains, being less dense due to their hydrated polysaccharide layer, localize near the top.
Weakly capsulated strains settle in the middle, and non-capsulated, denser strains migrate to the bottom.
The separated bacterial strains are ready for downstream analysis.
Carefully add 600 microliters of the prepared cells to the top of the gradient in the tube. Then place the tube in a tube adapter, and weigh the tube with the adapter to ensure balance. After this, place the tube adapter in a fixed angle rotor within a bench-top centrifuge, and centrifuge the tube for 30 minutes at 3, 000 g's.
Finally, after centrifugation, remove the tube carefully, place it in a rack, and photograph the results.
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