Team:LZU-China/wetlab

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       <td width="49%"><p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In wet lab, we designed a novel MFC device containing genetic engineered bacteria. For anode strain, we&rsquo;ve cloned a PNP sensor sequence and a riboflavin into Escherichia coli. The recombinants is able to detect PNP and produce riboflavin  to boost electrical generation when co-cultured with<em> Shewanella oneidensis. </em></p></td>
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        <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We constructed a pollutant substrate(PNP) bio-sensor coupling riboflavin synthetic gene cluster, the genetically modified E.coli can secrete riboflavin when added PNP in MFC anode medium.Riboflavin is a efficient redox mediator as well as a stimulator of MFC. We created a novel MFC devices and built a quantitative monitor system of PNP via measuring voltage increment.</p>
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<p><img src="https://static.igem.org/mediawiki/2014/4/40/Dht1_%281%29.jpg" width="238" height="75" /><a href="https://2014.igem.org/Team:LZU-China/wetlab2"><img src="https://static.igem.org/mediawiki/2014/2/2c/Dht1_(2).jpg" width="229" height="75" /></a><a href="https://2014.igem.org/Team:LZU-China/wetlab3"><img src="https://static.igem.org/mediawiki/2014/e/e8/Dht1_(3).jpg" width="204" height="75" /></a><a href="https://2014.igem.org/Team:LZU-China/wetlab4"><img src="https://static.igem.org/mediawiki/2014/0/02/Dht1_(4).jpg" width="299" height="75" /></a></p></td>
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      <div> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GENETIC ENGINEERED BACTERIA</div>
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            <td background="https://static.igem.org/mediawiki/2014/a/a6/Huils.jpg"  height="50">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;INTRODUCTION</td>
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             <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We designed this pathway for our purpose. As you can see, the NsrR is constitutive expressed in almost all E. coli orgnisms and it can repress the promoter PyeaR. In our project we found that this sensitive system is also adjusted to the PNP(p-nitrophenol). So we constructed this sensor pathway.</p>
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             <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Why did we choose the MFC as our device? MFC is an abbreviation of microbial fuel cell.MFC is a bioreactor that can convert biomass energy into electricity through the metabolic activity of bacteria in the anode chamber.(<a href="#_ENREF_1" title="Du, 2007 #10">Du,  Li, &amp; Gu, 2007</a>) . Nowadays, MFC is a new energy source available for us to solve pollution problem and produce electricity energy simultaneously. </p>
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            <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The sensor will cause the expression of riboflavin, a kind of mediator which can induce the generation of current in MFCs, to show the appearance of PNP. We can estimate the concentration of PNP. </p>
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            <p><img src="WETAhttps://static.igem.org/mediawiki/2014/e/ee/WETAhttps://static.igem.org/mediawiki/2014/e/ee/WETAimage001.png" width="800" height="467" /></p>
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            <p align="center">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Figure-1 The pathway of PNP sensor</p>
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             <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Besides, we find many genes can catalyze the reduction of Cr(VI). They can be used in the cathode to reduce these heavy metal ions.</p>
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             <p><img src="https://static.igem.org/mediawiki/2014/4/47/MFC_sketch.png" width="555" height="479" /></p>
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             <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<img src="WETAhttps://static.igem.org/mediawiki/2014/3/39/WETAhttps://static.igem.org/mediawiki/2014/3/39/WETAimage002.png" width="556" height="307" /></p>
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            <p align="center">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Figure-2 The pathway of the Cr ion reductase(yieF as an example)</p></td>
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              &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;However,  the main challenge of MFC is how to bring these technologies out of the laboratory  and engineer practical systems for bioenergy production at larger scales. One  of the most severe problems is the low efficiency of extracellular electron  transfer without the help of redox mediators added manually into  the reactor, such as neutral red, thionin (<a href="#_ENREF_5" title="Rabaey, 2005 #50">Rabaey  &amp; Verstraete, 2005</a>) . The addition of these mediators seemed to be another troublesome pollutant to deal with.<br />
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            <p><img src="https://static.igem.org/mediawiki/2014/1/1c/Duxuejing_cartoon_Bhalf.png" alt="" width="627" height="304" align="middle" /></p>
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            <p>&nbsp;</p>
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            <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In  our study, we successfully constructed a power stimulating model to solve this puzzle. We use <em>Shewanella</em>,  a well-known star of electricity-producing bacteria family, as our devices&rsquo;  powerful engine, it can send forth bacterial  <em>nanowires and reach the highest power output peak</em>. Furthermore, we construct an extraordinary &ldquo;<em>Turbo Boost</em>&rdquo; device&mdash;genetically  modified E.coli system&mdash;to stimulate this process. The hero of this story is riboflavin, a natural redox mediator also  known as Vitamin B2. Our E.coli system can produce  a certain amount of riboflavin, which concentration is dependent on substrate&rsquo;s  quantity, using a pollutant substrate bio-sensor coupling riboflavin synthetic  gene cluster.            </p>
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            <p><img src="https://static.igem.org/mediawiki/2014/6/6e/Old_MFC_photo.png" alt="" width="455" height="340" /></p>
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            <p>&nbsp; </p>
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            <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In that system, we can monitor pollutant concentration  real-timely by measuring the voltage of MFC devices and demonstrate result via data processing center and mobile network application.</p>
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            <p align="left">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<a href="https://2014.igem.org/Team:LZU-China/wetlab2"><img src="https://static.igem.org/mediawiki/2014/2/2c/L3.png" alt="" width="349" height="81" /></a></p>
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      <td ><div>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ABOUT THE CONSTRUCTION OF PNP SENSOR</div></td>
 
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        <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We found that the part BBa_K381001 by  iGEM10_BCCS-Bristol is a sensor which can detect the appearance of nitrate and  nitrite. So we perfomed an experience by this part to see if this part can  detect PNP(p-Nitrophenol).
 
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          We got a good result.You can see the PNP  can also induce the green fluorescence.</p>
 
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      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2014/3/3c/Image005.png" width="455" height="340" /></p>
 
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      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Figure-3 Fluorescence  of different system. </p>
 
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      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;a.bacterial liquid with 10mM PNP;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;b.bacterial liquid with  ddH2O;</p>
 
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      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;c.bacterial liquid with 10mM KNO3;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;d.bacterial  liquid with 10mM KCl.</p>
 
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      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Firstly we got the plasmid with K1172303  from Registry. We cut the plasmid by EcoRI and XbaI, then put the <em>Pnsr</em>(K216005+B0030  this sequence was synthesized by company)  into the gap.</p>
 
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      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<img src="image008.jpg" width="489" height="90" /></p>
 
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      <p align="center">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Figure-4  Construction of K1523101</p>
 
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      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The size of the whole part is about  3700bp(without plasmid), we test the assembling result by PCR(sense:5&rsquo;---TTCCCATCTATAATCCTCCCTGATTCTTCG---3&rsquo;;anti-sense:5&rsquo;---GAATTCTCTAGATTACAACTGTTGTTCAAGCTGTT---3&rsquo;).  From the gel picture we can see the size is right.</p>
 
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      <p align="center">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Figure-5  Gel picture of K1523101&rsquo;s PCR</p>
 
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      <td background="https://static.igem.org/mediawiki/2014/a/a6/Huils.jpg" ><div>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ABOUT THE REDUCING Cr IONS GENES</div></td>
 
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        <p align="left">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We found  four genes to reduce the Cr(VI) to Cr(III). They are<em> nahG, nahR, nahE, chrR</em> and <em>yieF. </em>We didn&rsquo;t do the deep research about them because of time  limitation. They can be used in the general plasmids such as pBR322. We just  made them to be the formation of Biobrick. More uses and functions need to be  demonstrated and found.</p>
 
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        <p align="left"><strong><em>nahG</em></strong>: We got the sequence from <em>Pseudomonas putida</em> </p>
 
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        <p align="left">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(Primer:sense:5&rsquo;---CCGGAATTCGCGGCCGCTTCTAGATGAAAAACAATAAACCTGGCTTGCGC---3&rsquo;;<br />
 
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          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;anti-sense:5&rsquo;---AAAATACTAGTAGCGGCCGCTGCAGTCACCCTTGACGTAGCACACC---3&rsquo;)<br />
 
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  <strong><em>nahE: </em></strong>We got the sequence from <em>Pseudomonas putida<strong> </strong></em><br />
 
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          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(Primer:sense:5&rsquo;---CCGGAATTCGCGGCCGCTTCTAGATGTCGAATAAAATTATGAAAACGTCGCG---3&rsquo;;<br />
 
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          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;anti-sense:5&rsquo;---AAAATACTAGTAGCGGCCGCTGCAGCTACTTCAATTCATTACTGTATTTAGCGTG---3&rsquo;)<br />
 
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  <strong><em>nahR: </em></strong>We got the sequence from <em>Pseudomonas putida<strong> </strong></em><br />
 
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          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(Primer:sense:5&rsquo;---  CCGGAATTCGCGGCCGCTTCTAGATGAAAAACAATAAACCTGGCTTGCGC---3&rsquo;;<br />
 
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          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;anti-sense:5&rsquo;---AAAATACTAGTAGCGGCCGCTGCAGTCACCCTTGACGTAGCACACC---3&rsquo;)<br />
 
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  <strong><em>chrR: </em></strong>We got the sequence from <em>Escherichia coli str.  K-12<strong> </strong></em><br />
 
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          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(Primer:sense:5&rsquo;---  CCGGAATTCGCGGCCGCTTCTAGATGAAAAAAATAGTCCAGTCGGAAGG---3&rsquo;;<br />
 
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          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;anti-sense:5&rsquo;---AAAATACTAGTAGCGGCCGCTGCAGTCAGGCCTTTTTGTGCTGTTCAAC---3&rsquo;)<br />
 
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  <strong><em>yieF: </em></strong>We got the sequence from <em>Escherichia coli str.  K-12<strong> </strong></em><br />
 
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          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(Primer:sense:5&rsquo;---  GAATTCGGATCCATGTCTGAAAAATTGCAGGTGG---3&rsquo;;<br />
 
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Latest revision as of 21:46, 17 October 2014

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" " http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> LZU-China 2014

 
 

 

 

 

         We constructed a pollutant substrate(PNP) bio-sensor coupling riboflavin synthetic gene cluster, the genetically modified E.coli can secrete riboflavin when added PNP in MFC anode medium.Riboflavin is a efficient redox mediator as well as a stimulator of MFC. We created a novel MFC devices and built a quantitative monitor system of PNP via measuring voltage increment.

 

 

  

 

 

 

 

       INTRODUCTION

 

             

         Why did we choose the MFC as our device? MFC is an abbreviation of microbial fuel cell.MFC is a bioreactor that can convert biomass energy into electricity through the metabolic activity of bacteria in the anode chamber.(Du, Li, & Gu, 2007) . Nowadays, MFC is a new energy source available for us to solve pollution problem and produce electricity energy simultaneously.

 


         However, the main challenge of MFC is how to bring these technologies out of the laboratory and engineer practical systems for bioenergy production at larger scales. One of the most severe problems is the low efficiency of extracellular electron transfer without the help of redox mediators added manually into the reactor, such as neutral red, thionin (Rabaey & Verstraete, 2005) . The addition of these mediators seemed to be another troublesome pollutant to deal with.

 

         In our study, we successfully constructed a power stimulating model to solve this puzzle. We use Shewanella, a well-known star of electricity-producing bacteria family, as our devices’ powerful engine, it can send forth bacterial nanowires and reach the highest power output peak. Furthermore, we construct an extraordinary “Turbo Boost” device—genetically modified E.coli system—to stimulate this process. The hero of this story is riboflavin, a natural redox mediator also known as Vitamin B2. Our E.coli system can produce a certain amount of riboflavin, which concentration is dependent on substrate’s quantity, using a pollutant substrate bio-sensor coupling riboflavin synthetic gene cluster.

 

         In that system, we can monitor pollutant concentration real-timely by measuring the voltage of MFC devices and demonstrate result via data processing center and mobile network application.

 

                                                                                                                

 

 

 

 

 

 

 

 

 

 

 

 

 

 

          

  

 

 

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