Difference between revisions of "Part:BBa K802001"

 
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<partinfo>BBa_K802001 short</partinfo>
 
<partinfo>BBa_K802001 short</partinfo>
  
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The part contains the DspB gene coding for an enzyme which catalyzes the hydrolysis of the extracellular matrix produced by Gram-negative bacteria.
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This part associates the <i>Bacillus subtillis</i> <i>Constitutive Promoter</i> (PVeg) with the <i>dispersin B</i> gene (DspB).The DspB gene code for an enzyme which catalyzes the hydrolysis of the extracellular matrix produced by Gram-negative bacteria.
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<br/>
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== Characterization ==
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<html>
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<p>Following results show that this part allows <i>B. subtillis</i> 168 strains to scatter the <i>S. aureus</i> and <i>epidermidis</i> cells in a biofilm. </p><br/>
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<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. subtillis</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 subtillis</i>.</p><br/>
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<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>
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<br/><br/><br/>
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<p> <font color="green" size="3">
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            Confocal Microscopy <a href="https://static.igem.org/mediawiki/2012/9/95/Tests_on_Staphylococcus_aureus_biofilms.pdf"><img src="https://static.igem.org/mediawiki/2011/6/64/Fileicon-pdf.png" style="width:25px";> </a><br><HR>
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          </font>
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      </p><br/>
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<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 biofilm in 96-well microscopic-grade microtiter plate.<br><br/>
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<i>Bacillus subtillis</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>
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After 24h of culture at 30°C without shaking, biofilm were observed under a time-lapse confocal microscope.<br>
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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>
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The 3D constructions are obtained with IMARIS software.</p>
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<p style="text-align:center"><big><b><i>S.aureus</i> biofilm no treated (Blank)</b></big></p>
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<p style="text-align:center"><big><b><i>S.aureus</i> biofilm treats by the strain with the shuttle vector without DspB gene (Negativ control)</b></big></p>
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<p style="text-align:center"><big><b><i>S.aureus</i> biofilm treats by the strain with the part</b></big></p>
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<p> <font color="green" size="3">
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            SDS-PAGE Protein gel  <br><HR>
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          </font>
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      </p><br/>
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<br/> <br/>
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</html>
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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This part was designed to be used.......
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Revision as of 21:03, 21 September 2012

Dispersin generator for B. subtilis

This part associates the Bacillus subtillis Constitutive Promoter (PVeg) with the dispersin B gene (DspB).The DspB gene code 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. subtillis 168 strains to scatter the S. aureus and epidermidis cells in a biofilm.


In our plasmid collection, this part is named pBK33 in the backbone Chloramphenicol and pBKH41 in the shuttle vector E. coli – B. subtillis. 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 E. coli and the strain 168 to make test in Bacillus subtillis.


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.


Confocal Microscopy


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

Bacillus subtillis 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).
After 24h of culture at 30°C without shaking, biofilm were observed under a time-lapse confocal microscope.
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 are obtained with IMARIS software.


S.aureus biofilm no treated (Blank)


S.aureus biofilm treats by the strain with the shuttle vector without DspB gene (Negativ control)


S.aureus biofilm treats by the strain with the part

SDS-PAGE Protein gel





Usage and Biology

This part was designed to be used.......


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]