Difference between revisions of "Part:BBa K1597002"

Revision as of 14:52, 18 September 2015

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1597002 short</partinfo>
-''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.
+''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 P''proH''promoter  <html><a href="https://parts.igem.org/Part:BBa_K1597000"> (BBa_K1597000) </a></html> before ''tasA'' before integration in ''B. subtilis''. This construct was integrated in ''b. subtilis'' genome with the use of <html><a href="https://parts.igem.org/Part:BBa_K823023"> BBa_K823023 </a></html>, resulting in a ''tasA'' mutant. With this construct was desinged to induce the production of TasA protein with different salt concentration.
+In our construct we added the salt inducible P''proH''promoter  <html><a href="https://parts.igem.org/Part:BBa_K1597000"> (BBa_K1597000) </a></html> before ''tasA'' before integration in ''B. subtilis''. This construct was integrated in the ''B. subtilis'' genome with the use of <html><a href="https://parts.igem.org/Part:BBa_K823023"> BBa_K823023 </a></html>, resulting in a ''tasA'' overexpression strain.
-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.
+The ''tasA'' and wild type ''B. subtilis NCIB'' 3610 ComI (Δ''comI'') 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 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.
+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.
@@ Line 15: / Line 16: @@
 </html>
-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 <html><a href="https://parts.igem.org/Part:BBa_K1597000"> (BBa_K1597000) </a></html> and the ''tasA'' biobrick function in ''B. subtilis''.
+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 <html><a href="https://parts.igem.org/Part:BBa_K1597000"> (BBa_K1597000) </a></html> and the ''tasA'' biobrick function in ''B. subtilis''.