Part:BBa_K1597002
tasA, amyloid-like fibers
tasA is teh gene 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 synthesized. These fibers attach to the cell wall. Together with extracellular polysaccharides amyloid fibers promote formationof cell clusters in the form of bundles of cel chains. This 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 the B. subtilis genome with the use of BBa_K823023 , resulting in a tasA overexpression strain.
The tasA and wild type B. subtilis NCIB 3610 ComI (ΔcomI) were The tasA and B. subtilis ComI (comI) were grown on Msgg media with or without salt. After 24 and 48 hours 50 mM thioflavin S (which is an amyloid fiber stain) 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 not the case for the wild type strain. After 48 hours the differences are less visible, which could be explained by 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 overexpression strain. 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 wildtype B. subtilis and in the tasA overexpression strain. Both strains were grown on SSM media (supplemented with 10 µM thioflavin S) with different salt concentrations, ranging from 0M NaCl up to 1 M NaCl. Over time the fluorescence (em: 430 nm, ex: 485 nm, Gain 50) and the OD (600 nm) were measured using a plate reader. The plates were incubated 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 the wildtype and tasA overexpression strain. After one hour the tasA overexpression strain show a higher fluorescence than the control. After 3 hours the tasA overexpression strain 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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