Curli Fiber-Mediated Stabilization of Bacterial Hydrogel Patterns

Take two dishes containing 3D-printed alginate matrices, each encapsulating either control or test bacterial strains.

Both strains constitutively express a fluorescent protein for visualization. The test strain also contains an inducible gene for producing proteinaceous extracellular fibers called curli fibers.

The patterns are printed on a solid medium supplemented with calcium and rhamnose.

Incubate to allow calcium ions to crosslink the alginate, forming a hydrogel.

In the test strain, rhamnose induces the production of curli subunits, which self-assemble at the cell surface into fibers.

Use a fluorescent scanner to image the patterns.

Treat with a calcium-chelating solution and incubate with shaking to sequester calcium ions, which disrupts the alginate crosslinks and dissolves the hydrogel.

Discard the solution and re-image the patterns.

The treatment caused the control pattern to dissolve, while the test pattern remained intact due to the stabilizing network of curli fibers.

Incubate the printed samples at room temperature for three to six days to allow the production of the biofilm components, such as Curli fibers.

Then, place the plate on a fluorescent scanner and image the plates.

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