Difference between revisions of "Part:BBa K802001"

Latest revision as of 22:25, 26 September 2012

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 Staphyloccocus epidermids 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

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 to provide the swimmers "biofilm killer".

We verify the inocuity of our constructs on B. subtilis. With or without vector we found that B. subtilis was able to swarm the plate overnight. Our tests also show that B. subtilis is a much better swimmer than E. coli.

Motility plate assays

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.
Biofilms with or without addition of B. subtilis were then observed under a time-lapse confocal microscope as described (see protocol). GFP was excited at 488 nm with an argon laser, and fluorescent emission was collected on a detector in the range of 500-600 nm. Biofilms were observed by 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 : untreated S. aureus biofilm.

Negative control : S. aureus biofilm treated with B. subtilis containing the shuttle vector without the dspB gene.

S. aureus biofilm treated for 4 hours with B. subtilis containing the part BBa_K802001 carried by the shuttle vector.

Conclusion :

Our results demonstrate that the part BBa K802001 leads to the almost complete S. aureus biofilm dispersal

Quantitative image analysis :

A quantitative analysis was performed 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 were analysed.

Conclusion :

The results obtained from the blank and the negative control show that if washing doesn't affect the biofilm when the cells are not first scattered by the action of dispersin, S. aureus biofilm treated with B. subtilis containing the part BBa K802001 is significantly reduced after washing.

^[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 Staphylococcus epidermidis biofilm thickness after Dispersin treatment

In order to measure and to appreciate the Dispersin effect on Staphylococcus epidermidis biofilms, tests were conducted in two different systems : in microtiter plates and on glass lamella in test tubes.

Microtiter plates : Each well was inoculated with a 1:100 dilution of a 24h TSB Staphyloccocus epidermids preculture. From crystal violet staining, we can easily see that untreated S. epidermidis biofilms adhere to the well walls. The adherence is estimated from OD₆₀₀ to be ca. 75%. However the Staphylococcus epidermidis biofilm on polystyren or glass coverslip was found too fragile for the manipulation required to test the dispersin effect.

The same kind of tests were done in test tubes containing coverslip. The results are similar to those done on microtiter plates biofilms is destroyed even in presence of the negative control.

OD₆₀₀ Test

We measured OD₆₀₀ to demonstrate the effect of dispersin on S. epidermidis. This test shows that dispersin has a limited effect on a bacterial suspension of S. epidermidis. This result was expected since dispersin only scatters bacterial aggregates and thus does not reduce the number of cells which is measured when we take OD₆₀₀.

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

General conclusion

Our results clearly show the dramatic increase in efficiency of Biofilm Killer, which expresses the lysostaphin and dispersin parts in combination, over the other Bacillus subtilis strains harboring only the lysostaphin or dispersin parts.

Usage and Biology

This part was designed to be used in Bacillus strains in order to scatter biofilms including Staphyloccocus aureus biofilm which we have used here.
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]

@@ Line 7: / Line 7: @@
 <html>
-<p>Following results show that the BBa K802001 part allows <i>B. subtilis</i> 168 strains to scatter <i>S. aureus</i> cells in a biofilm. </p><br/>
+<p>Following results show that the BBa K802001 part allows <i>B. subtilis</i> 168 strains to scatter <i>S. aureus</i> and <i>Staphyloccocus epidermids</i> cells in a biofilm. </p><br/>
 <p>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 <i>E. coli</i> – <i>B. subtilis</i>. The corresponding negative control is the empty shuttle vector (pBKH26 in our collection). The plasmid pBKH41 was introduced into both <i>E. coli</i>  NM522 and <i>Bacillus subtilis</i> 168 strains to assay its scattering performance.</p><br/>
@@ Line 19: / Line 19: @@
        </p><br/>
-<i><b>Bacillus subtilis</i> 168</b> was transformed with pBKH41 (<i>dspB</i> 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 to provide the swimmers "biofilm killer".<br>
+<i><b>Bacillus subtilis</i> 168</b> was transformed with pBKH41 (<i>dspB</i> 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 to provide the swimmers "biofilm killer".<br>
+<p>We verify the inocuity of our constructs on <i>B. subtilis</i>. With or without vector we found that <i>B. subtilis</i> was able to swarm the plate overnight. Our tests also show that <i>B. subtilis</i> is a much better swimmer than <i>E. coli</i>.
+</br>
+<p style="text-align:center"><big><b>Motility plate assays</b></big></p>
+<div style="text-align:center">
+<img src="https://static.igem.org/mediawiki/2012/8/83/Mobile.jpg" width="600px">
+</div>
+</br></br>
 <p><b>Biofilms of the <i>S. aureus</i> fluorescent strain</b> RN4220 pALC2084(GFP-tagged) were cultivated in 96-wells microtiter plates. This strain is a nonmotile laboratory strain.<br>Biofilms with or without addition of <i>B. subtilis</i> were then observed under a time-lapse confocal microscope as described (see protocol). GFP was excited at 488 nm with an argon laser, and fluorescent emission was collected on a detector in the range of 500-600 nm. Biofilms were observed by 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.</p> <b>For each well, two observations were made : one before washing the biofilm and one after washing.</b><br>
@@ Line 47: / Line 54: @@
 <br/>
-<p style="text-align:center"><big><b><i>S. aureus</i> biofilm treated with <i>B. subtilis</i> containing the part BBa_K802001 carried by the shuttle vector.</b>
+<p style="text-align:center"><big><b><i>S. aureus</i> biofilm treated for 4 hours with <i>B. subtilis</i> containing the part BBa_K802001 carried by the shuttle vector.</b>
 </big></p>
 <div style="text-align:center">
@@ Line 54: / Line 61: @@
 </br></br>
-<div style="font-size:19px;font-weight:bold;width:80%;text-align:center;margin:auto;"> Conclusion : Our results demonstrate that the part BBa K802001 leads to the almost complete <i>S. aureus</i> biofilm dispersal</div>
+<div style="width:80%;text-align:center;margin:auto;"> <div style="font-size:19px;font-weight:bold;">Conclusion :<br></div><br> Our results demonstrate that the part BBa K802001 leads to the almost complete <i>S. aureus</i> biofilm dispersal</div>
 </br></br>
@@ Line 69: / Line 76: @@
 </br></br>
 <div style="text-align:center">
-<img src="https://static.igem.org/mediawiki/2012/d/d0/Total_biovolume_of_S.aureu.jpg" width="600px">
+<img src="https://static.igem.org/mediawiki/2012/c/c1/Graph2disp.jpg" width="600px">
 </div>
 </br></br>
 <div style="text-align:center">
-<img src="https://static.igem.org/mediawiki/2012/6/66/Substratum_coverage.jpg" width="600px">
+<img src="https://static.igem.org/mediawiki/2012/4/4c/Graph1disp.jpg" width="600px">
 </div>
 </br></br>
-<div style="font-size:19px;font-weight:bold;width:80%;text-align:center;margin:auto;"> Conclusion:
+<div style="width:80%;text-align:center;margin:auto;"> <div style="font-size:19px;font-weight:bold;">Conclusion :<br></div>
 </br> The results obtained from the blank and the negative control show that if washing doesn't affect the biofilm when the cells are not first scattered by the action of dispersin, <i>S. aureus</i> biofilm treated with <i>B. subtilis</i> containing the part BBa K802001 is significantly reduced after washing.</div>
 </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.
@@ Line 82: / Line 89: @@
 <p> <font color="green" size="3">
-              Quantification of biofilm thickness after Dispersin treatment <a href="'https://static.igem.org/mediawiki/2012/5/55/Tests_on_Staphylococcus_epidermidis_biofilms_in_24_wells_plate.pdf"><img src="https://static.igem.org/mediawiki/2011/6/64/Fileicon-pdf.png" style="width:25px";> </a><br><HR>
+              Quantification of <i>Staphylococcus epidermidis</i> biofilm thickness after Dispersin treatment <a href="'https://static.igem.org/mediawiki/2012/5/55/Tests_on_Staphylococcus_epidermidis_biofilms_in_24_wells_plate.pdf"><img src="https://static.igem.org/mediawiki/2011/6/64/Fileicon-pdf.png" style="width:25px";> </a><br><HR>
            </font>
        </p><br/>
-<p>In order to measure and to appreciate the Dispersin effect on <i> Staphylococcus epidermidis </i> biofilms, tests on microtiter plates, test tubes and lamella  were leaded. </p>
+<p>In order to measure and to appreciate the Dispersin effect on <i>Staphylococcus epidermidis</i> biofilms, tests were conducted in two different systems : in microtiter plates and on glass lamella in test tubes. </p>
-<p>Each well was inoculated with a <i> Staphyloccocus epidermids </i> 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 <i> S. epidermidis biofilms </i> adhere to well's walls and by measuring the D.O. at 600 nm ,the adherence is estimated to be around 75%.</p>
+<p>Microtiter plates : Each well was inoculated with a 1:100 dilution of a 24h TSB <i>Staphyloccocus epidermids</i> preculture. From crystal violet staining, we can easily see that untreated <i>S. epidermidis</i> biofilms adhere to the well walls. The adherence is estimated from OD<sub>600</sub> to be ca. 75%.
+However the <i>Staphylococcus epidermidis</i> biofilm on polystyren or glass coverslip was found too fragile for the manipulation required to test the dispersin effect.</p>
-<div style="text-align:center">
+<p>The same kind of tests were done in test tubes containing coverslip. The results are similar to those done on microtiter plates biofilms is destroyed even in presence of the negative control. </p>
-<img src="https://static.igem.org/mediawiki/2012/5/52/IMG_0854.JPG" width="250px">
-</div>
-<p>After a 24-hour incubation of the biofilm with a <i> Bacillus subtilis </i> supernatant containing or not Dispersin (negative control), the biofilm is detached. Indeed, <i> S. epidermidis </i> 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.  </p>
-<p>The same kind of tests were done but in test tubes containing a lamella. </p>
-<p>The test tube with the lamella in is inoculated with 5 mL suspension obtained from a culture of <i> S. epidermidis </i> diluted 1:100 with TSB supplemented with glucose 1%. After 24 h the supernatant is replaced with a <i> B. subtilis </i> 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).
-</p>
-<p>In conclusion, <i>S. epidermidis</i> biofilms are not the best model to test the Dispersin effect on biofilms.
-Other experiences have demonstrated better results were tests were done on <i> S. aureus </i> biofilms. </p>
+</br></br>
+<p> <font color="green" size="3">
+             OD<sub>600</sub> Test  <a href="https://static.igem.org/mediawiki/2012/2/25/0D%28600%29_Test.pdf"><img src="https://static.igem.org/mediawiki/2011/6/64/Fileicon-pdf.png" style="width:25px";> </a><br><HR>
+          </font>
+      </p><br/>
+<p>We measured OD<sub>600</sub> to demonstrate the effect of dispersin on <i>S. epidermidis</i>. This test shows that dispersin has a limited effect on a bacterial suspension of <i>S. epidermidis</i>. This result was expected since dispersin only scatters bacterial aggregates and thus does not reduce the number of cells which is measured when we take OD<sub>600</sub>.
+</br></br>
 <p> <font color="green" size="3">
               SDS-PAGE Protein gel  <a href="https://static.igem.org/mediawiki/2012/9/96/SDS-PAGE_Protocol.pdf"><img src="https://static.igem.org/mediawiki/2011/6/64/Fileicon-pdf.png" style="width:25px";> </a><br><HR>
@@ Line 140: / Line 141: @@
 </div>
 </br></br>
+<center><big><b>General conclusion</b></big></center>
+<p>
+Our results clearly show the dramatic increase in efficiency of Biofilm Killer, which expresses the lysostaphin and dispersin parts in combination, over the other <i>Bacillus subtilis</i> strains harboring only the lysostaphin or dispersin parts.
+</br></br></br>
 </html>
@@ Line 145: / Line 151: @@
 ===Usage and Biology===
-This part was designed to be used in <i>Bacillus</i> strains in order to scatter a <i>Staphyloccocus aureus</i> biofilm.
+This part was designed to be used in <i>Bacillus</i> strains in order to scatter biofilms including <i>Staphyloccocus aureus</i> biofilm which we have used here.
 <br>Possible applications include biofilm treatment in medical domain, oil or food-processing industries, using this scattering property to eliminate harmful biofilms.