Team:UNITN-Trento/Project/Bacillus

From 2013.igem.org

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Bacillus Subtilis
Bacillus Subtilis
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When we first came up with the idea of B- fruity, we immediatly thought that b.subtilis was the perfect chassis for a possible marketable application:
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When we first came up with the idea of <i>B. fruity</i>, we immediatly thought that <i>B .subtilis</i> was the perfect chassis for a possible marketable application:
<ol>
<ol>
<li>
<li>
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</li>
</li>
<li>
<li>
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<i>Bacillus subtilis</i> is not pathogenic;
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<i>Bacillus subtilis</i> is not pathogenic and therefore can be used safely for food applications.;
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we have always worked only with <i>E. coli</i> and we thought to try out how it is to employ a new organism. It’s been really challenging but we got it!
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</li>
</li>
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</ol>
</ol>
<i>Bacillus subtilis</i> would be the perfect chassis for a fruit-ripening household product, that exploit ethylene (or MeSA) production upon spores activation. We have designed a <i>B. fruity</i> home edition that exploits this principle. <br/>
<i>Bacillus subtilis</i> would be the perfect chassis for a fruit-ripening household product, that exploit ethylene (or MeSA) production upon spores activation. We have designed a <i>B. fruity</i> home edition that exploits this principle. <br/>
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To achieve this goal we started working with EFE, a ethylene forming enzyme from <i>Pseudomas Syringae</i> pv. phaseolicola (<a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>), which were inserted into pSBBs0K-Pspac (IPGT inducible) and  pSBBs4S-Pxyl (xylose inducible), two biobrick plasmids designed for <i>B. subtilis</i> by the iGEM 2012 LMU Munich team (please note that we used a new functional version of these plasmids, that were sent to us from LMU Munich).
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To achieve this goal we started working with EFE, a ethylene forming enzyme from <i>Pseudomas Syringae</i> pv. phaseolicola (<a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>), which were inserted into pSBBs0K-Pspac (IPGT inducible) and  pSBBs4S-Pxyl (xylose inducible), two biobrick plasmids designed for <i>B. subtilis</i> by the iGEM 2012 LMU Munich team (please note that we used a new functional version of these plasmids, that were kindly sent to us from LMU Munich).
<img src="http://2013.igem.org/wiki/images/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/>
<img src="http://2013.igem.org/wiki/images/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/>
<span class="tn-subtitle">Cloning of BBa_K1065203</span>
<span class="tn-subtitle">Cloning of BBa_K1065203</span>
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The integrative plasmid pXyl was digested prior transformation in minimal media and the correct integration of the insert into B. subtilis genome was confirmed with the threonine assay.
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The integrative plasmid pXyl was digested prior transformation in minimal media and the correct integration of the insert into <i>B. subtilis</i> genome was confirmed with the threonine assay.
<div class="tn-doublephoto-wrap">
<div class="tn-doublephoto-wrap">
<img class="photo" src="http://2013.igem.org/wiki/images/b/b0/Tn-2013_PXyl_digestion.png"/>
<img class="photo" src="http://2013.igem.org/wiki/images/b/b0/Tn-2013_PXyl_digestion.png"/>
<img class="photo" src="http://2013.igem.org/wiki/images/3/3b/Tn-2013_thr_assay.jpg"/>
<img class="photo" src="http://2013.igem.org/wiki/images/3/3b/Tn-2013_thr_assay.jpg"/>
</div>
</div>
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<span class="tn-caption"><b>Figure 1:</b> transformation of <a href="http://parts.igem.org/Part:BBa_K1065203">BBa_1065203</a> in <i>B. subtilis</i>. Transformation of the integrative vector pXyl carrying the EFE gene was achieved by digesting the plasmid with ScaI to obtain a linear DNA (left panel) which was then transformed into <i>B. subtilis</i> 168 using minimal medium.  Correct integration was confirmed with the threonince test: cells that carry the insert in the proper position become auxotrophic and can not longer grow in the absence of threonine.</span>
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<span class="tn-caption"><b>Figure 1:</b> transformation of <a href="http://parts.igem.org/Part:BBa_K1065203">BBa_1065203</a> in <i>B. subtilis</i>. Transformation of the integrative vector pXyl carrying the EFE gene was achieved by digesting the plasmid with ScaI to obtain a linear DNA (left panel) which was then transformed into <i>B. subtilis</i> 168 using minimal medium.  Correct integration was confirmed with the threonine test: cells that carry the insert in the proper position become auxotrophic and can not longer grow in the absence of threonine.</span>
<span class="tn-subtitle">Toxicity assay</span>
<span class="tn-subtitle">Toxicity assay</span>
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We then measured the optical density of cells induced and non induced for both contructs.
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We then measured the optical density of cells induced and non induced for both constructs.
<div class="tn-doublephoto-wrap">
<div class="tn-doublephoto-wrap">
<img class="plot" src="http://2013.igem.org/wiki/images/8/8e/Tn-2013_K1065203_plot.png">
<img class="plot" src="http://2013.igem.org/wiki/images/8/8e/Tn-2013_K1065203_plot.png">
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Ethylene production was tested by Gas Chromatography as we previoulsy did for <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>.  The experiment was performed both from cultures started from fresh plates and from dry spores.<br/>
Ethylene production was tested by Gas Chromatography as we previoulsy did for <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>.  The experiment was performed both from cultures started from fresh plates and from dry spores.<br/>
Unfortunately, we did not observe any production of ethylene after 4 hours, nor after overnight induction. <br/>
Unfortunately, we did not observe any production of ethylene after 4 hours, nor after overnight induction. <br/>
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At this point we are not able to confirm that EFE was correctly expressed under these conditions. Surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips . Hydrogen and sulfate react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip.  
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At this point we are not able to confirm that EFE was correctly expressed under these conditions. Surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips . Hydrogen sulfide and other mercapto compounds react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip.  
<img src="http://2013.igem.org/wiki/images/6/69/Tn-2013_Lead-acetate_strip_assay.jpg"/>
<img src="http://2013.igem.org/wiki/images/6/69/Tn-2013_Lead-acetate_strip_assay.jpg"/>
<span style="text-align:justify;"class="tn-caption center"><b>Figure 4:</b> detection of sulfur compounds. <i>B. subtilis</i> 168 cells non transformed (1 and 2), transformed with <a href="http://2013.igem.org/wiki/images/8/8e/Tn-2013_K1065203_plot.png">BBa_K1065203</a> (3 and 4) and transformed with <a href="http://2013.igem.org/wiki/images/8/8e/Tn-2013_K1065204_plot.png">BBa_K1065204</a> (5 and 6) were grown until O.D. 0.8 was reached. Samples 1 and 4 were then supplemented with 1% xyloyse while samples 2 and 5 were supplemented with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples (4 and 5) showed a darker strip indicating the presence of sulfur compounds. The non trasformed cells supplemented with the inducer did not show that precipitate. </span>
<span style="text-align:justify;"class="tn-caption center"><b>Figure 4:</b> detection of sulfur compounds. <i>B. subtilis</i> 168 cells non transformed (1 and 2), transformed with <a href="http://2013.igem.org/wiki/images/8/8e/Tn-2013_K1065203_plot.png">BBa_K1065203</a> (3 and 4) and transformed with <a href="http://2013.igem.org/wiki/images/8/8e/Tn-2013_K1065204_plot.png">BBa_K1065204</a> (5 and 6) were grown until O.D. 0.8 was reached. Samples 1 and 4 were then supplemented with 1% xyloyse while samples 2 and 5 were supplemented with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples (4 and 5) showed a darker strip indicating the presence of sulfur compounds. The non trasformed cells supplemented with the inducer did not show that precipitate. </span>

Revision as of 20:35, 30 September 2013

Bacillus Subtilis When we first came up with the idea of B. fruity, we immediatly thought that B .subtilis was the perfect chassis for a possible marketable application:
  1. Bacillus subtilis sporulates and it can be stored in a inactive state;
  2. Bacillus subtilis is not pathogenic and therefore can be used safely for food applications.;
Bacillus subtilis would be the perfect chassis for a fruit-ripening household product, that exploit ethylene (or MeSA) production upon spores activation. We have designed a B. fruity home edition that exploits this principle.
To achieve this goal we started working with EFE, a ethylene forming enzyme from Pseudomas Syringae pv. phaseolicola (BBa_K1065002), which were inserted into pSBBs0K-Pspac (IPGT inducible) and pSBBs4S-Pxyl (xylose inducible), two biobrick plasmids designed for B. subtilis by the iGEM 2012 LMU Munich team (please note that we used a new functional version of these plasmids, that were kindly sent to us from LMU Munich). Cloning of BBa_K1065203 The integrative plasmid pXyl was digested prior transformation in minimal media and the correct integration of the insert into B. subtilis genome was confirmed with the threonine assay.
Figure 1: transformation of BBa_1065203 in B. subtilis. Transformation of the integrative vector pXyl carrying the EFE gene was achieved by digesting the plasmid with ScaI to obtain a linear DNA (left panel) which was then transformed into B. subtilis 168 using minimal medium. Correct integration was confirmed with the threonine test: cells that carry the insert in the proper position become auxotrophic and can not longer grow in the absence of threonine. Toxicity assay We then measured the optical density of cells induced and non induced for both constructs.
Figure 2: B. subtilis 168 cells transformed with BBa_K1065203 or BBa_K1065204 were grown until an OD=0.9 and then splitted in two samples before induction. Cells were induced with 1% xylose for BBa_K1065203 and 0.5 mM of IPTG for BBa_K1065204. In both cases the induced samples grow slightly slower than the controls. Sporulation assay Spores were obtained by growing the transformed B. subtilis 168 cells in DSM medium, subjecting them to a heat shock at 60 °C and plating them on a preheated glass slide. Spores were visualized at the microscope. Figure 3: B. subtilis spores shown with 100X zoom. Ethylene detection Ethylene production was tested by Gas Chromatography as we previoulsy did for BBa_K1065001. The experiment was performed both from cultures started from fresh plates and from dry spores.
Unfortunately, we did not observe any production of ethylene after 4 hours, nor after overnight induction.
At this point we are not able to confirm that EFE was correctly expressed under these conditions. Surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips . Hydrogen sulfide and other mercapto compounds react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip. Figure 4: detection of sulfur compounds. B. subtilis 168 cells non transformed (1 and 2), transformed with BBa_K1065203 (3 and 4) and transformed with BBa_K1065204 (5 and 6) were grown until O.D. 0.8 was reached. Samples 1 and 4 were then supplemented with 1% xyloyse while samples 2 and 5 were supplemented with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples (4 and 5) showed a darker strip indicating the presence of sulfur compounds. The non trasformed cells supplemented with the inducer did not show that precipitate.