Difference between revisions of "Part:BBa K1597002"
 
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''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 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.
  
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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 ''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.
 +
 
 +
Phenotypical differences from ''tasA'' overproduction strain, grown on Msgg with (0,5M) and without NaCl, are described <html><a href="https://static.igem.org/mediawiki/parts/7/76/TasA_and_bslA_single_and_double_mutant.pdf"> tasA and bslA single and double overproduction strains</a></html>.  
  
 
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  ''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.  
+
With the induction of salt, ''tasA'' shows more amyloid fibers are present after 24 hours. This is not 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.  
  
  
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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 ''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''
  
  
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<img src="https://static.igem.org/mediawiki/parts/9/96/Tecan.jpg"
 
<img src="https://static.igem.org/mediawiki/parts/9/96/Tecan.jpg"
 
</html>
 
</html>
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 +
To validate if our project could actually work a prototype was developed where the different strains could be tested on the chosen carrier. First, different strains were tested with two types of water; 30 g/L NaCl and 1g/L NaCl. This was done to recreate the effect of salt and fresh water, where a theoretical max of 86 mV could be achieved. <i>B. subtilis tasA</i>  achieved 16,5 mV energy potential  Another tested setup was <i>B. subtilis</i> with an overproduction of <i> tasA,bslA</i>, mixed with <i>B. subtilis ΔabrB slrR</i>.  This mixture gave a 17,5 mV.
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[[File:prototype.jpg]]
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Although these test showed that an energy potential could be generated, a real-world prototype was not yet demonstrated. This could be achieved with the use of real seawater and water from a fresh lake(movie). Due to time limits only the <i>B. subtilis</i> with an overproduction of tasA, <i>bslA</i>, mixed with <i>B. subtilis</i> ΔabrB slrR was measured with these circumstances. This measurement resulted in a 21,5 mV energy potential, which is 25% of the theoretically maximum. In the movie the amount of mV is visible on the multimeter. The flow cell can also be seen (link naar flow cell), where there are two compartments, one with fresh and one with salt water. In between of these compartments a biofilm can be put in. Our engineered <i>B. subtilis</i> strains are capable of generating a energy potential over the membrane while simulating the actual conditions in the Netherlands. Please see the video below!
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[[Media:Prototype sea, lake.mp4.ogg]]
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 04:18, 19 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.

Phenotypical differences from tasA overproduction strain, grown on Msgg with (0,5M) and without NaCl, 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 not 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


To validate if our project could actually work a prototype was developed where the different strains could be tested on the chosen carrier. First, different strains were tested with two types of water; 30 g/L NaCl and 1g/L NaCl. This was done to recreate the effect of salt and fresh water, where a theoretical max of 86 mV could be achieved. B. subtilis tasA achieved 16,5 mV energy potential Another tested setup was B. subtilis with an overproduction of tasA,bslA, mixed with B. subtilis ΔabrB slrR. This mixture gave a 17,5 mV.

Prototype.jpg

Although these test showed that an energy potential could be generated, a real-world prototype was not yet demonstrated. This could be achieved with the use of real seawater and water from a fresh lake(movie). Due to time limits only the B. subtilis with an overproduction of tasA, bslA, mixed with B. subtilis ΔabrB slrR was measured with these circumstances. This measurement resulted in a 21,5 mV energy potential, which is 25% of the theoretically maximum. In the movie the amount of mV is visible on the multimeter. The flow cell can also be seen (link naar flow cell), where there are two compartments, one with fresh and one with salt water. In between of these compartments a biofilm can be put in. Our engineered B. subtilis strains are capable of generating a energy potential over the membrane while simulating the actual conditions in the Netherlands. Please see the video below!

Media:Prototype sea, lake.mp4.ogg


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]