Part:BBa_K802001

Dispersin generator for B. subtilis

This part associates the Bacillus subtilis constitutive promoter (P_veg) 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 the BBa K802001 part allows B. subtilis 168 strains to scatter S. aureus and S. epidermidis cells in a biofilm.

In our plasmid collection, this part is refered to pBK33 when cloned into the pSB1C3 (CmR) backbone, and pBKH41 when cloned into the high copy shuttle vector E. coli – B. subtilis. The corresponding negative control is the empty shuttle vector (pBKH26 in our collection). The plasmid pBKH41 was introduced into both E. coli NM522 and Bacillus subtilis 168 strains to assay its scattering performance.

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 of the S. aureus fluorescent strain RN4220 pALC2084(GFP-tagged)were cultivated in 96-wells microtiter plates. This strain is a nonmotile laboratory strain.

Bacillus subtilis 168 was transformed with pBKH41 (dspB in the shuttle vector) and with pBKH26 (empty shuttle vector as a negative control). These two strains were grown on LB medium supplemented with erythromycin (15µg/mL)for 24h at 30°C without shaking. Biofilms were then observed under a time-lapse confocal microscope as described in the protocol. 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.

For each well, two observations were made : one before washing the biofilm and one after washing.

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.

Quantification of biofilm thickness after Dispersin treatment

In order to measure and to appreciate the Dispersin effect on Staphylococcus epidermidis biofilms, tests on microtiter plates, test tubes and lamella were leaded.

Each well was inoculated with a Staphyloccocus epidermids culture grown for 24 hours in TSB and diluted 1:100 with TSB supplemented with 1% glucose. After incubation and crystal violet coloration, it can easily be seen that S. epidermidis biofilms adhere to well's walls and by measuring the D.O. at 600 nm ,the adherence is estimated to be around 75%.

After a 24-hour incubation of the biofilm with a Bacillus subtilis supernatant containing or not Dispersin (negative control), the biofilm is detached. Indeed, S. epidermidis biofilms are very sensible to medium growth changes and the fact of not using TSB for supernatant tests, does not allow the experimentator to conclude whether or not the biofilms destruction is the result of Dispersin action.

The same kind of tests were done but in test tubes containing a lamella.

The test tube with the lamella in is inoculated with 5 mL suspension obtained from a culture of S. epidermidis diluted 1:100 with TSB supplemented with glucose 1%. After 24 h the supernatant is replaced with a B. subtilis supernatant containing or not Dispersin(negative control). But, the results are similar to those done on microtiter plates: the biofilms is destroyed even in presence of the negative control (data not shown).

In conclusion, S. epidermidis biofilms are not the best model to test the Dispersin effect on biofilms. Other experiences have demonstrated better results were tests were done on S. aureus biofilms.

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:

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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
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COMPATIBLE WITH RFC[1000]