Part:BBa_K802001
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. For each well, two observations are 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 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 675
- 1000COMPATIBLE WITH RFC[1000]
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