Difference between revisions of "Part:BBa K802001"
Line 2: Line 2:
 
<partinfo>BBa_K802001 short</partinfo>
 
<partinfo>BBa_K802001 short</partinfo>
  
−
This part associates the <i>Bacillus subtilis</i> <i>Constitutive Promoter</i> (PVeg) with the <i>dispersin B</i> gene (DspB).The DspB gene codes for an enzyme which catalyzes the hydrolysis of the extracellular matrix produced by Gram-negative bacteria.
+
This part associates the <i>Bacillus subtilis</i> <i>Constitutive Promoter</i> (P<sub>veg</sub>) with <i>dispersin B</i> gene (<i>dspB</i>).The <i>dspB</i> gene codes for an enzyme which catalyzes the hydrolysis of the extracellular matrix produced by Gram negative bacteria.
 
<br/>
 
<br/>
 
== Characterization ==
 
== Characterization ==
 
<html>
 
<html>
  
−
<p>Following results show that this part allows <i>B. subtilis</i> 168 strains to scatter the <i>S. aureus</i> and <i>epidermidis</i> cells in a biofilm. </p><br/>
+
<p>Following results show that this part allows <i>B. subtilis</i> 168 strains to scatter <i>S. aureus</i> and <i>S. epidermidis</i> cells in a biofilm. </p><br/>
  
−
<p>In our plasmid collection, this part is named pBK33 in the backbone Chloramphenicol and pBKH41 in the shuttle vector <i>E. coli</i> – <i>B. subtilis</i>. The corresponding negative control is the shuttle vector (pBKH26 in our collection). We worked with the plasmid pBKH41 for the tests and we tried two different genetic backgrounds: the strain NM522 to make test in <i>E. coli</i> and the strain 168 to make test in <i>Bacillus subtilis</i>.</p><br/>
+
<p>In our plasmid collection, this part is named pBK33 in the Chloramphenicol backbone and pBKH41 in the shuttle vector <i>E. coli</i> – <i>B. subtilis</i>. The corresponding negative control is the shuttle vector (pBKH26 in our collection). We worked with the plasmid pBKH41 for the tests and we tried two different genetic backgrounds : NM522 strain to make test in <i>E. coli</i> and 168 strain to make test in <i>Bacillus subtilis</i>.</p><br/>
  
−
<a href="http://2012.igem.org/Team:Lyon-INSA/protocol"/><font color="grey"><b>In you have any question on the following experiments, don’t forget that all the informations relative to our strains, plasmids and protocols are on our wiki notebook.</b></font></a>
+
<a href="http://2012.igem.org/Team:Lyon-INSA/protocol"/><font color="grey"><b>If you have any question on the following experiments, don’t forget that all the informations relative to our strains, plasmids and protocols are on our wiki notebook.</b></font></a>
 
<br/><br/><br/>
 
<br/><br/><br/>
  
Line 20: Line 20:
  
 
<p>Biofilms are formed by the <i>S. aureus</i> fluorescent strain RN4220 pALC2084 expressing GFP. It is a nonmotile laboratory strain, used to form biofilms in 96-well microscopic-grade microtiter plate.<br><br/>
 
<p>Biofilms are formed by the <i>S. aureus</i> fluorescent strain RN4220 pALC2084 expressing GFP. It is a nonmotile laboratory strain, used to form biofilms in 96-well microscopic-grade microtiter plate.<br><br/>
−
<i>Bacillus subtilis</i> 168 transformed by pBKH41 (DspB in in the shuttle vector) and by pBKH26 (the shuttle vector without gene to have a negative control) were grown on LB medium supplemented with erythromycin (15µg/mL).<br>  
+
<i>Bacillus subtilis</i> 168 transformed with pBKH41 (<i>dspB</i> in the shuttle vector) and with pBKH26 (shuttle vector without gene to have a negative control) were grown on LB medium supplemented with erythromycin (15µg/mL).<br>  
 
After 24h of culture at 30°C without shaking, biofilms were observed under a time-lapse confocal microscope. <b>For each well, two observations were made : one before washing the biofilm and one after washing (i.e. after removing the supernatant).</b><br>  
 
After 24h of culture at 30°C without shaking, biofilms were observed under a time-lapse confocal microscope. <b>For each well, two observations were made : one before washing the biofilm and one after washing (i.e. after removing the supernatant).</b><br>  
 
Cells expressing GFP were excited at 488 nm with an argon laser, and fluorescent emission was collected on a detector in the range of 500-600 nm using an oil-immersion objective with a magnification of 63x. The overall three-dimensional structures of the biofilms were scanned from the solid surface to the interface with the growth medium, using a step of 1 µm.<br>
 
Cells expressing GFP were excited at 488 nm with an argon laser, and fluorescent emission was collected on a detector in the range of 500-600 nm using an oil-immersion objective with a magnification of 63x. The overall three-dimensional structures of the biofilms were scanned from the solid surface to the interface with the growth medium, using a step of 1 µm.<br>
Line 26: Line 26:
 
</br>
 
</br>
 
Three cases are analysed :
 
Three cases are analysed :
−
</br><ul><b>-Blank</b> : it is a non treated <i>S. aureus</i> biofilm (just with growth medium).
+
</br><ul>
−
</br><b>-Negative control</b> : it is a <i>S. aureus</i> biofilm treated by <i>B. subtilis</i> containing the shuttle vector without the DspB gene.
+
<li><b>Blank</b> : it is a non treated <i>S. aureus</i> biofilm (just with growth medium).</li>
−
</br><b>-Strain with our part</b> : it is a <i>S. aureus</i> biofilm treated by <i>B. subtilis</i> containing the part BBa_K802001 in the shuttle vector.
+
<li><b>Negative control</b> : it is a <i>S. aureus</i> biofilm treated with <i>B. subtilis</i> containing the shuttle vector without the <i>dspB</i> gene.</li>
 +
<li><b>Strain with our part</b> : it is a <i>S. aureus</i> biofilm treated with <i>B. subtilis</i> containing the part BBa_K802001 in the shuttle vector.</li>
 +
</ul>
 
</br></br>
 
</br></br>
 
<br/>  
 
<br/>  
Line 38: Line 40:
  
 
<br/>  
 
<br/>  
−
<p style="text-align:center"><big><b><i>S.aureus</i> biofilm treated by the strain with the shuttle vector without DspB gene (Negative control)</b></big></p>
+
<p style="text-align:center"><big><b><i>S.aureus</i> biofilm treated with the strain containing the shuttle vector without <i>dspB</i> gene (Negative control)</b></big></p>
 
<div style="text-align:center">
 
<div style="text-align:center">
 
<img src="https://static.igem.org/mediawiki/2012/b/ba/S.aureus_biofilm_without_control_treatment_before_and_after_washing.jpg" width="600px">  
 
<img src="https://static.igem.org/mediawiki/2012/b/ba/S.aureus_biofilm_without_control_treatment_before_and_after_washing.jpg" width="600px">  
Line 45: Line 47:
  
 
<br/>  
 
<br/>  
−
<p style="text-align:center"><big><b><i>S.aureus</i> biofilm treated by the strain with the part</b></big></p>
+
<p style="text-align:center"><big><b><i>S.aureus</i> biofilm treated with the strain containing the part</b></big></p>
 
<div style="text-align:center">
 
<div style="text-align:center">
 
<img src="https://static.igem.org/mediawiki/2012/2/2e/S.aureus_biofilm_with_dispersin_treatment_before_and_after_washing.jpg" width="600px">  
 
<img src="https://static.igem.org/mediawiki/2012/2/2e/S.aureus_biofilm_with_dispersin_treatment_before_and_after_washing.jpg" width="600px">  
Line 51: Line 53:
 
</br>
 
</br>
  
−
<big><b>Conclusion:</b></big>
+
<big><b>Conclusion :</b></big>
  
−
</br> With these observations, we concluded that the part allowed <b>to scatter a <i>S. aureus</i> biofilm after washing</b>. The bindings between the <i>S. aureus</i> cells inside the biofilm are affected, so that the biofilm can be easily eliminated after washing.   
+
</br> With these observations, we concluded that the part enabled <b>to scatter a <i>S. aureus</i> biofilm after washing</b>. The bounds between <i>S. aureus</i> cells inside the biofilm are affected, so that the biofilm can be easily eliminated after washing.   
 
</br></br></br>
 
</br></br></br>
  
−
<big><b><h4>Statistic analysis:</h5></b></big>
+
<big><b><h4>Statistic analysis :</h5></b></big>
−
</br>In order to quantify our results, we made a statistical analysis with the MATLAB software. Different parameters [1] were used to quantify the biofilm, particularly :
+
</br>In order to quantify our results, we made a statistical analysis with the MATLAB software. Different parameters<sup>[1]</sup> were used to quantify the biofilm, particularly :
−
</br><ul><b>- Total Biovolume (µm<sup>3</sup>)</b> : it corresponds to the overall volume of the biofilm and also allows to have an estimation of the biomass in the biofilm.
+
</br><ul>
−
</br><b>- Substratum coverage (%)</b> : it corresponds to the area coverage in the first image of the stack (i.e. at the substratum). It is a good mean to estimate how efficiently the substratum is colonized by bacteria of the population.
+
<li><b>Total Biovolume (µm<sup>3</sup>)</b> : it corresponds to the overall volume of the biofilm and also allows to have an estimation of the biomass in the biofilm.</li>
 +
<li><b>Substratum coverage (%)</b> : it corresponds to the area coverage in the first image of the stack (i.e. at the substratum). It is a good mean to estimate how efficiently the substratum is colonized by bacteria of the population.</li>
 +
</ul>
 
</br></br>
 
</br></br>
 
The same three cases as previously are analysed.
 
The same three cases as previously are analysed.
Line 73: Line 77:
 
</br></br>
 
</br></br>
 
<big><b>Conclusion:</b></big>
 
<big><b>Conclusion:</b></big>
−
</br>These statistical results demonstrate that the <i>S. aureus</i> biofilm treated by the <i>B. subtilis</i> with the part is significantly reduced after washing. The blank and the negative control prove that washing doesn't affect the biofilm when the cells are not first scattered by the action of dispersin.
+
</br>These statistical results demonstrate that <i>S. aureus</i> biofilm treated with <i>B. subtilis</i> containing the part is significantly reduced after washing. The blank and the negative control prove that washing doesn't affect the biofilm when the cells are not first scattered by the action of dispersin.
−
</br></br>[1]Quantification of biofilm structures by the novel computer program COMSTAT. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S.Molecular Microbial Ecology Group, Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark.July 2000.
+
</br></br><sup>[1]</sup>Quantification of biofilm structures by the novel computer program COMSTAT. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S.Molecular Microbial Ecology Group, Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark.July 2000.
 
</br></br></br></br>
 
</br></br></br></br>
 
<p> <font color="green" size="3">
 
<p> <font color="green" size="3">
Line 80: Line 84:
 
           </font>
 
           </font>
 
       </p><br/>
 
       </p><br/>
−
     No massive production of dispersin could be observed in <i>E. coli</i> or in <i>B. subtilis</i> supernatant, even after a 4X concentration by acetone precipitation (data not shown.)       
+
     No massive production of dispersin could be observed in <i>E. coli</i> nor in <i>B. subtilis</i> supernatant, even after a 4X concentration by acetone precipitation (data not shown).      
  
 
<br/> <br/>
 
<br/> <br/>
Line 90: Line 94:
 
<p>As we demontrated with the previous tests, the part BBa_K802001 has a real effect on the <i>S. aureus</i> biofilm. For our Biofilm Killer project, two <b>complementary</b> agents (lysostaphin with the part BBa_K802000 and dispersin with the part BBa_K802001) were used to destroy an installed biofilm. Thus, it was interesting for us to combine these two agents.  
 
<p>As we demontrated with the previous tests, the part BBa_K802001 has a real effect on the <i>S. aureus</i> biofilm. For our Biofilm Killer project, two <b>complementary</b> agents (lysostaphin with the part BBa_K802000 and dispersin with the part BBa_K802001) were used to destroy an installed biofilm. Thus, it was interesting for us to combine these two agents.  
 
<br>
 
<br>
−
We also have made new tests on 96 wells plate, according to the same protocole than the one used to characterize the lysostaphin part with the confocal microscope. The only difference was that we add 125µL of <i>B. subtilis</i> with the part BBa_K802000 and 125µL of <i>B. subtilis</i> with the part BBa_K802001.  
+
We have also made new tests on 96-well plate, according to the same protocole than the one used to characterize the lysostaphin part with the confocal microscope. The only difference was that we added 125µL of <i>B. subtilis</i> with the part BBa_K802000 and 125µL of <i>B. subtilis</i> with the part BBa_K802001.  
 
</br>
 
</br>
 
Two cases are analysed :
 
Two cases are analysed :
−
</br><ul><b>- Negative control</b> : it is a <i>S. aureus</i> biofilm treated by <i>B. subtilis</i> strains containing the shuttle vectors without the Lysostaphin and Dispersin genes.
+
</br><ul>
−
</br><b>- Strain with our parts</b> : it is a <i>S. aureus</i> biofilm treated by <i>B. subtilis</i> strains containing the part BBa_K802000 and BBa_K802001 in their shuttle vectors.
+
<li><b>Negative control</b> : it is a <i>S. aureus</i> biofilm treated with <i>B. subtilis</i> strains containing the shuttle vectors without the Lysostaphin and Dispersin genes.</li>
 +
<li><b>Strain with our parts</b> : it is a <i>S. aureus</i> biofilm treated with <i>B. subtilis</i> strains containing the part BBa_K802000 and BBa_K802001 in their shuttle vectors.</li>
 +
</ul>
 
</br></br>
 
</br></br>
  
 
<br/>  
 
<br/>  
−
<p style="text-align:center"><big><b><i>S.aureus biofilm</i> treated by the shuttle vectors without the lysostaphin and the dispersin genes (Negative control)</b></big></p>
+
<p style="text-align:center"><big><b><i>S.aureus</i> biofilm treated with the shuttle vectors without the lysostaphin and the dispersin genes (Negative control)</b></big></p>
 
<div style="text-align:center">
 
<div style="text-align:center">
 
<img src="https://static.igem.org/mediawiki/2012/3/3f/Effet_combin%C3%A9_%28t%C3%A9moin%29.jpg" width="600px">  
 
<img src="https://static.igem.org/mediawiki/2012/3/3f/Effet_combin%C3%A9_%28t%C3%A9moin%29.jpg" width="600px">  
 
</div>
 
</div>
 
</br></br>
 
</br></br>
−
<p style="text-align:center"><big><b><i>S.aureus biofilm</i> treated by the strain with the Parts BBa_K802000 and BBa_K802001 </b></big></p>
+
<p style="text-align:center"><big><b><i>S.aureus</i> biofilm treated with the strain containing parts BBa_K802000 and BBa_K802001 </b></big></p>
 
<div style="text-align:center">
 
<div style="text-align:center">
 
<img src="https://static.igem.org/mediawiki/2012/5/5a/S.aureus_lyso%2Bdisp.jpg" width="600px">  
 
<img src="https://static.igem.org/mediawiki/2012/5/5a/S.aureus_lyso%2Bdisp.jpg" width="600px">  

Revision as of 00:08, 26 September 2012

Dispersin generator for B. subtilis

This part associates the Bacillus subtilis Constitutive Promoter (Pveg) with dispersin B gene (dspB).The dspB gene codes for an enzyme which catalyzes the hydrolysis of the extracellular matrix produced by Gram negative bacteria.

Characterization

Following results show that this part allows B. subtilis 168 strains to scatter S. aureus and S. epidermidis cells in a biofilm.


In our plasmid collection, this part is named pBK33 in the Chloramphenicol backbone and pBKH41 in the shuttle vector E. coli – B. subtilis. The corresponding negative control is the shuttle vector (pBKH26 in our collection). We worked with the plasmid pBKH41 for the tests and we tried two different genetic backgrounds : NM522 strain to make test in E. coli and 168 strain to make test in Bacillus subtilis.


If you have any question on the following experiments, don’t forget that all the informations relative to our strains, plasmids and protocols are on our wiki notebook.


Confocal Microscopy


Biofilms are formed by the S. aureus fluorescent strain RN4220 pALC2084 expressing GFP. It is a nonmotile laboratory strain, used to form biofilms in 96-well microscopic-grade microtiter plate.

Bacillus subtilis 168 transformed with pBKH41 (dspB in the shuttle vector) and with pBKH26 (shuttle vector without gene to have a negative control) were grown on LB medium supplemented with erythromycin (15µg/mL).
After 24h of culture at 30°C without shaking, biofilms were observed under a time-lapse confocal microscope. For each well, two observations were made : one before washing the biofilm and one after washing (i.e. after removing the supernatant).
Cells expressing GFP were excited at 488 nm with an argon laser, and fluorescent emission was collected on a detector in the range of 500-600 nm using an oil-immersion objective with a magnification of 63x. The overall three-dimensional structures of the biofilms were scanned from the solid surface to the interface with the growth medium, using a step of 1 µm.
The 3D constructions were obtained with IMARIS software.


Three cases are analysed :
  • Blank : it is a non treated S. aureus biofilm (just with growth medium).
  • Negative control : it is a S. aureus biofilm treated with B. subtilis containing the shuttle vector without the dspB gene.
  • Strain with our part : it is a S. aureus biofilm treated with B. subtilis containing the part BBa_K802001 in the shuttle vector.



S.aureus biofilm not treated (Blank)




S.aureus biofilm treated with the strain containing the shuttle vector without dspB gene (Negative control)




S.aureus biofilm treated with the strain containing the part


Conclusion :
With these observations, we concluded that the part enabled to scatter a S. aureus biofilm after washing. The bounds between S. aureus cells inside the biofilm are affected, so that the biofilm can be easily eliminated after washing.


Statistic analysis :
In order to quantify our results, we made a statistical analysis with the MATLAB software. Different parameters[1] were used to quantify the biofilm, particularly :
  • Total Biovolume (µm3) : it corresponds to the overall volume of the biofilm and also allows to have an estimation of the biomass in the biofilm.
  • Substratum coverage (%) : it corresponds to the area coverage in the first image of the stack (i.e. at the substratum). It is a good mean to estimate how efficiently the substratum is colonized by bacteria of the population.


The same three cases as previously are analysed.





Conclusion:
These statistical results demonstrate that S. aureus biofilm treated with B. subtilis containing the part is significantly reduced after washing. The blank and the negative control prove that washing doesn't affect the biofilm when the cells are not first scattered by the action of dispersin.

[1]Quantification of biofilm structures by the novel computer program COMSTAT. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S.Molecular Microbial Ecology Group, Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark.July 2000.



SDS-PAGE Protein gel


No massive production of dispersin could be observed in E. coli nor in B. subtilis supernatant, even after a 4X concentration by acetone precipitation (data not shown).

Combined action between the parts BBa_K802000 and BBa_K802001


As we demontrated with the previous tests, the part BBa_K802001 has a real effect on the S. aureus biofilm. For our Biofilm Killer project, two complementary agents (lysostaphin with the part BBa_K802000 and dispersin with the part BBa_K802001) were used to destroy an installed biofilm. Thus, it was interesting for us to combine these two agents.
We have also made new tests on 96-well plate, according to the same protocole than the one used to characterize the lysostaphin part with the confocal microscope. The only difference was that we added 125µL of B. subtilis with the part BBa_K802000 and 125µL of B. subtilis with the part BBa_K802001.
Two cases are analysed :

  • Negative control : it is a S. aureus biofilm treated with B. subtilis strains containing the shuttle vectors without the Lysostaphin and Dispersin genes.
  • Strain with our parts : it is a S. aureus biofilm treated with B. subtilis strains containing the part BBa_K802000 and BBa_K802001 in their shuttle vectors.



S.aureus biofilm treated with the shuttle vectors without the lysostaphin and the dispersin genes (Negative control)



S.aureus biofilm treated with the strain containing parts BBa_K802000 and BBa_K802001




Usage and Biology

This part was designed to be used in Bacillus strains in order to scatter a Staphyloccocus aureus biofilm.
Possible applications include biofilm treatment in medical domain, oil or food-processing industries, using this scattering property to eliminate harmful biofilms.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 675
  • 1000
    COMPATIBLE WITH RFC[1000]