Difference between revisions of "Part:BBa K1597002"

Revision as of 15:57, 18 September 2015

tasA, amyloid-like fibers

tasA is responsible for the amyloid-like fibers that are one of the main constituents of the extracellular matrix in Bacillus subtilis biofilms. After the expression of the tapA-sipW-tasA operon, TasA proteins (amyloid-like fibres) are synthesised. These fibers attach to the cell wall. Together with extracellular polysaccharides, tasA forms amyloid-like fibers promoting formation of cell clusters, resemble=ing bundles of cell chains. This effect results in a more robust biofilm.

In our construct we added the salt inducible PproHpromoter (BBa_K1597000) before tasA before integration in B. subtilis. This construct was integrated in b. subtilis genome with the use of BBa_K823023 , resulting in a tasA mutant. With this construct was desinged to induce the production of TasA protein with different salt concentration.

The tasA overproduction strain was grown on Msgg without NaCl and on Msgg on 0,5M NaCl. Phenotical differences are described tasA and bslA single and double overproduction strains.

The tasA and B. subtilis ComI (comI) were grown on Msgg media with or without salt. After 24 and 48 hours thioflavin S (which is an amyloid fiber staining) was added to the biofilms. After 15 min incubation at room temperature the biofilms were photographed with white light and fluorescence.

With the induction of salt, tasA shows more amyloid fibers are present after 24 hours. This is nTt the case for comI. After 48 hours the differences are less visible, which could be explained bu the fact that tasA is a natural gene in B. subtilis which is expressed in a later phase of biofilm production. The natural gene is not yet fully activated after 24 hours, where it is with salt induction in the tasA mutant. This means that the salt inducible PproH promoter is activated with salt and produces extra TasA protein.

Another method is to measure TasA protein with thioflavin S over time in both B. subtilis comI and the tasA mutant. Both strains were grown on SSM media (supplemented with 10 µM thioflavin S) with different salt concentrations, ranging from 0M salt up to 1 M salt. Over time the fluorescence and the OD were measured. The plates were incubated during measurement an in between at 37 °C. Strains grown on a salt concentration above 0,5M showed little growth. Therefore these results are not shown here. A bar chart was plotted after 1 hour and after 3 hours for both B. subtilis comI and tasA mutant. After one hour the tasA mutant show a higher fluorescence than the B. subtilis comI control. After 3 hours the tasA mutant shows, with an induction of 0,1M; 0,2M and 0,3M salt, a significant increase than the control. This confirms that both the salt-inducible (BBa_K1597000) and the tasA biobrick function in B. subtilis.

Sequence and Features