Team:UNITN-Trento/Project/Bacillus

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			<i>Bacillus subtilis</i>
		</span>
		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>
			<li>
				<i>Bacillus subtilis</i> sporulates and can be stored in an inactive state;
			</li>	
			<li>
				<i>Bacillus subtilis</i> is not pathogenic and therefore can be used safely for food applications.
			</li>
		</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 designed a <i>B. fruity</i> home edition based on this principle.<br />
		To achieve this goal we started working with EFE, an 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) These two bio-brick plasmids were designed for <i>B. subtilis</i> by the iGEM 2012 LMU Munich team (please note that we used the new functional versions of these plasmids that were kindly provided by LMU Munich).
		<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/>
		
		<span class="tn-subtitle">Cloning of BBa_K1065204</span>
		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 by threonine assay.
		<div class="tn-doublephoto-wrap">
			<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b0/Tn-2013_PXyl_digestion.png"/>
			<img class="photo" src="https://static.igem.org/mediawiki/2013/3/3b/Tn-2013_thr_assay.jpg"/>
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		<span class="tn-caption"><b>Figure 1:</b> transformation of <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_1065204</a> in <i>B. subtilis</i>. Transformation of the integrative vector pXyl carrying the EFE gene was achieved by digestion of the plasmid with ScaI to obtain a linear DNA (left panel) which was then transformed into <i>B. subtilis</i> 168 cultured in minimal medium.  Correct integration was confirmed by threonine test: cells that carry the insert in the proper position become auxotrophic and can no longer grow in the absence of threonine.</span>
		
		<span class="tn-subtitle">Toxicity assay</span>
		We measured the optical density of cells induced and non induced for both constructs.
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		<img class="plot" src="https://static.igem.org/mediawiki/2013/1/14/Tn-2013_K1065204_plot.png">	
		<img class="plot" src="https://static.igem.org/mediawiki/2013/8/8e/Tn-2013_K1065203_plot.png">		
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		<span class="tn-caption"><b>Figure 2:</b> <i>B. subtilis</i> 168 cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_K1065204</a> or <a href="http://parts.igem.org/Part:BBa_K1065203">BBa_K1065203</a> were grown until an OD of 0.9 and then split in two samples before induction. Cells were induced with 1% xylose for BBa_K1065204 and 0.5 mM of IPTG for BBa_K1065203. In both cases the induced samples (blue) grow slightly slower than the controls (red).</span>

		<span class="tn-subtitle">Sporulation assay</span>
		Spores were obtained growing the transformed <i>B. subtilis</i> 168 cells in DSM medium, subjecting them to a heat shock at 60 &deg;C and plating them on a preheated glass slide. Spores were visualized at the microscope.

		<span class="tn-subtitle">Ethylene detection</span>
		Ethylene production was tested by Gas Chromatography as we previously 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/>
		We did not observe any production of ethylene after 4 hours, nor after overnight induction.<br/>
		At this point we are not able to confirm that EFE was correctly expressed under these conditions but, 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="https://static.igem.org/mediawiki/2013/6/69/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, transformed with <a href="http://parts.igem.org/Part:BBa_K1065203">BBa_K1065203</a> and transformed with <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_K1065204</a> were grown until O.D. 0.9 was reached. At this O.D. one sample was then supplemented with 1% xylose or with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples showed a darker strip indicating the presence of sulfur compounds. Non transformed cells supplemented with the inducer did not show that precipitate.</span>

		<span class="tn-subtitle">Future directions</span>
		For future experiments and improvement of the system we have identified additional potential drawbacks, including:
		<ul>
		<li>pXyl could be inhibited by glucose although the threonine test confirmed the correct insertion of the vector;</li>
		<li>the acquisition of pSpac could not be confirmed by colony PCR yet; even if  the growth of colonies in the presence of the  antibiotic indicates that the episomal vector carrying EFE gene is present;</li>
		<li>at present the expression of the EFE gene has not been demonstrated. We plan to perform an additional real-time PCR experiment (to assess transcription).</li>
		</ul> 
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