Difference between revisions of "Part:BBa K802009"

 
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<partinfo>BBa_K802009 short</partinfo>
 
<partinfo>BBa_K802009 short</partinfo>
  
 
This part can be used to induce surfactin production (an antimicrobial lipopeptide) and to repress the biofilm formation in B. subtilis strains.  
 
This part can be used to induce surfactin production (an antimicrobial lipopeptide) and to repress the biofilm formation in B. subtilis strains.  
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==Characterization==
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<html>
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<br>
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<p> <div style="font-weight:bold">abrB test</div> </p>
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<p>
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The part was transformed into the BSΔabrB B. subtilis strain containing the mutated biofilm repressor gene (abrB). As a result, this strain forms biofilms, so if the part is functional, the transformed strain should not form biofilms. In fact, the transformed BSΔabrB strain with BBa_K802009 should be similar to the wild-type B. subtilis BS168 strain  as far as the biofilm formation is concerned. </p>
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<br><p>
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In order to test the biofilm formation of the transformed bacteria, a qualitative test was performed in a 24-well microplate. Saturated liquid cultures were used to inoculate 2 mL of LB media (dilution ratio of the tested culture  compared to the saturated culture: 1/100) supplemented or not with 2% xylose. The negative control was the BS168ΔabrB strain (with and without xylose). The positive control was the wild-type B. subtilis BS168 strain
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(with and without xylose).
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<div style="text-align:center;style:left; margin-top:20px;margin-left:10px;">
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<img src="https://static.igem.org/mediawiki/2012/4/4f/2plaque.jpg" width="50%"/>
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</div>
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<div style="text-align:center;font-style>italic;color:blue">
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<p>
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the first row of wells does not contain xylose; the second row was supplemented with xylose 2%</p>
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</div>
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</p>
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<p>
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The results show that the characteristics of transformed mutated strain resemble more to the BS168 strain than to the BS168ΔabrB strain. Moreover, the two controls do not present a significant difference concerning biofilm formation between the xylose and xylose free media. As a result, the repression of biofilm formation is due to the part BBa_K802009.
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</p>
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<br><br>
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<br>
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<p> <div style="font-weight:bold">sfp test</div> </p>
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<p>
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In order to confirm the presence of the sfp gene, a qualitative test for surfactant production was performed. 2 mL of filtered supernatant coming from saturated cultures of transformed bacteria was mixed with 2 mL of cooking oil in a  spectrometer cuve. Afterwards, the cuve was thoroughly vortexed and the resulted emulsion was observed after 20 hours:  </p>
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<br>
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<div style="text-align:center;style:left; margin-top:20px;margin-left:10px;">
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<img src="https://static.igem.org/mediawiki/2012/e/ee/Tubes.jpg" width="50%"/>
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</div>
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<div style="text-align:center;font-style>italic;color:blue">
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<p>
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observed spectrophotometer cuves</p>
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</div>
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</p>
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<p>
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Sample number details:<br>
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  - 1 is a positive control (the supernatant was replaced with a 10% SDS solution);<br>
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  - 2 is a negative control (supernatant from the BS168 strain);<br>
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  - 3,4,5,6,7,8 contain the supernatant from tested clones;<br>
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  - 9 is a negative control (supernatant from BS168 transformed with the same plasmid which was used for cloning the part BBa_K802009, but without the part).<br>
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</p>
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<p>
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Comparing the cuves 9 and 2, it is safe to say that the observed effect of the supernatant is due to the part and not to the plasmid in which the part was cloned. Moreover, there is a significant difference between the tested clones and the negative control, so the part BBa_K802009 affects the emulsification capacity of the supernatant produced by the transformed bacteria.
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</p>
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<br><br>
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</html>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 02:51, 26 September 2012

Sufactin generator and biofilm repressor for B. subtilis

This part can be used to induce surfactin production (an antimicrobial lipopeptide) and to repress the biofilm formation in B. subtilis strains.


Characterization


abrB test

The part was transformed into the BSΔabrB B. subtilis strain containing the mutated biofilm repressor gene (abrB). As a result, this strain forms biofilms, so if the part is functional, the transformed strain should not form biofilms. In fact, the transformed BSΔabrB strain with BBa_K802009 should be similar to the wild-type B. subtilis BS168 strain as far as the biofilm formation is concerned.


In order to test the biofilm formation of the transformed bacteria, a qualitative test was performed in a 24-well microplate. Saturated liquid cultures were used to inoculate 2 mL of LB media (dilution ratio of the tested culture compared to the saturated culture: 1/100) supplemented or not with 2% xylose. The negative control was the BS168ΔabrB strain (with and without xylose). The positive control was the wild-type B. subtilis BS168 strain (with and without xylose).

the first row of wells does not contain xylose; the second row was supplemented with xylose 2%

The results show that the characteristics of transformed mutated strain resemble more to the BS168 strain than to the BS168ΔabrB strain. Moreover, the two controls do not present a significant difference concerning biofilm formation between the xylose and xylose free media. As a result, the repression of biofilm formation is due to the part BBa_K802009.




sfp test

In order to confirm the presence of the sfp gene, a qualitative test for surfactant production was performed. 2 mL of filtered supernatant coming from saturated cultures of transformed bacteria was mixed with 2 mL of cooking oil in a spectrometer cuve. Afterwards, the cuve was thoroughly vortexed and the resulted emulsion was observed after 20 hours:


observed spectrophotometer cuves

Sample number details:
- 1 is a positive control (the supernatant was replaced with a 10% SDS solution);
- 2 is a negative control (supernatant from the BS168 strain);
- 3,4,5,6,7,8 contain the supernatant from tested clones;
- 9 is a negative control (supernatant from BS168 transformed with the same plasmid which was used for cloning the part BBa_K802009, but without the part).

Comparing the cuves 9 and 2, it is safe to say that the observed effect of the supernatant is due to the part and not to the plasmid in which the part was cloned. Moreover, there is a significant difference between the tested clones and the negative control, so the part BBa_K802009 affects the emulsification capacity of the supernatant produced by the transformed bacteria.




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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 760
    Illegal SapI.rc site found at 770