Difference between revisions of "Part:BBa K1602017"
 
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         <h1>Xylose to xylitol converting construct</h1>
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         <h1><small>D</small>-xylonic acid producing operon</h1>
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        <b>Xylose</b> is a monosaccharide belonging to the aldopentose family. Through reduction it can be converted to xylitol. The reaction takes place in the cytosol of the host and recent studies show, that the formation of xylitol in E.coli seems possible as well.  
+
        <small>D</small>-Xylose is a monosaccharide belonging to the aldopentose family. It was recently    shown that the <small>D</small>-xylose dehydrogenase <i>xylB</i> from <i>Caulobacter crescentus</i> can convert <small>D</small>-xylose to <small>D</small>-xylonolactone. This can react spontaneously or through the catalysation of <i>xylC</i> to <small>D</small>-xylonic acid. (2)
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<br>
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In <i>E. coli</i> <small>D</small>-xylonic acid can further be metabolized to ethyleneglycol by the enzymes yjhG <a href="https://parts.igem.org/Part:BBa_K1602012">(BBa_K1602012)</a>, yagE <a href="https://parts.igem.org/Part:BBa_K1602011">(BBa_K1602011)</a> and yqhD <a href="https://parts.igem.org/Part:BBa_K1602013">(BBa_K1602013)</a> which are already present in this host. (1)
 
         <br>
 
         <br>
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         To enable the reduction in <i>E.coli</i> it is mandatory to establish an operon containing the coding gene <i>GRE3</i> for a aldose reductase. The gene is taken from <i>Saccharomyces cerevisiae</i>. The aldose reductase converts xylose to xylitol in dependence of NADPH.
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         <br>
 
         <br>
 
         <br>
 
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<div align="center">
 
<div align="center">
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         <img style="width: 565px; height: 110px; margin-left: 15px; margin-right: 15px;" alt="" src="https://static.igem.org/mediawiki/2015/7/7b/Conversion_xylose_xylitol_v1.png"></div>
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         <img class="shrinkToFit transparent" alt="https://static.igem.org/mediawiki/parts/f/f1/TU_Darmstadt_EG_XylB-xylC.png" src="https://static.igem.org/mediawiki/parts/f/f1/TU_Darmstadt_EG_XylB-xylC.png" height="407" width="169">
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        <p style="width: 900px; margin-left: 15px; margin-right: 15px;" alt="" text-align="left">
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             <b>Figure 1</b> Reaction scheme of the xylose to xylitol converting operon. Xylose is the only substrate needed for the reaction. Xylose is metabolized to xylitol in 1 step in dependance of NADPH.
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             <li><b>Figure 1</b> Sheme of the reactions catalyzed by xylB and xylC. The xylC reaction can also happen spontaneously but in a much lower speed. (2)</li>
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</div>
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</p>
 
         </p>
 
         </p>
 
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===<h2>Usage</h2>===
 
===<h2>Usage</h2>===
 
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             This part is a composite of one coding gene and a strong RBS (<a href="/Part:BBa_B0034">BBa_B0034</a>) in front of it, under control of a T7 Promoter (<a href="/Part:BBa_I719005">BBa_I719005</a>).
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             This part is a composite of two coding genes with strong RBS (<a href="/Part:BBa_B0034">BBa_B0034</a>). The transcription is controlled by a T7 promotor (<a href="/Part:BBa_I719005">BBa_I719005</a>).
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        <br>
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        <br>
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        We used this operon to investigate possible production of ethylene glycol in <i>E. coli</i>.
 
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                 <td>
 
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                     <ul>
 
                     <ul>
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                        <li class="block-10vi">aldose reductase - GRE3                     <a href="/Part:BBa_K1602004">(BBa_K1602004)</a></li>
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                    <li class="block-10vi">T7-promotor                      
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                    <a href="/Part:BBa_I719005">(BBa_I719005)</a></li>
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                    <li class="block-10vi">ribosome binding site B0034                    
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                    <a href="/Part:BBa_B0034">(BBa_B0034)</a></li>
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                    <li class="block-10vi"><small>D</small>-xylose dehydrogenase xylB                    
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                    <a href="/Part:BBa_K1602009">(BBa_K1602009)</a></li>
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 +
                    <li class="block-10vi"><small>D</small>-xylonolactone lactonase   
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xylC                    
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                    <a href="/Part:BBa_K1602010">(BBa_K1602010)</a></li>
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                     </ul>
 
                     </ul>
 
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                         <img style="width: 332px; height: 200px;" alt="" src="https://static.igem.org/mediawiki/2015/7/7e/T7_gre3_operon_v1.png" align="center">  
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                         <img class="transparent" alt="https://static.igem.org/mediawiki/parts/1/12/TU_Darmstadt_Plasmidkarte_T7-B0034-xylB-B0034-xylC.png" width="75%" height="75%" src="https://static.igem.org/mediawiki/parts/1/12/TU_Darmstadt_Plasmidkarte_T7-B0034-xylB-B0034-xylC.png">
 
                  
 
                  
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<div style="float: right;"><img style="width: 400px; height: 120px;" alt="" src="
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https://static.igem.org/mediawiki/2015/a/a1/Itaconic_acid_gene_operon.png"> </div>
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                     <b>Figure 2</b> Genetic map of the xylose to xylitol converting operon with T7 promoter. This brick enables <i>E.Coli</i> BL21 cells to convert xylose to xylitol in presence of the inductor IPTG.
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                 </td>
 
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     </body>
 
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</html>  
 
</html>  
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===<h2>Results</h2>===
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<html>
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<p align="justify"><i>E. coli</i> BL21 were transformed with the operon and grown to a OD of 0.6. A negative sample was taken before IPTG was added to a concentration of 1mM for induction. Cells stayed at 28°C for 12 hours and later were harvested and resuspended in buffer. A small amount of both induced samples and negative samples was loaded on a SDS-PAGE while proteins were extracted from the rest. The SDS-PAGE showed overexpression of proteins of the expected mass.</p>
 +
<p align="justify">In a NAD<sup>+</sup> assay activity of <i>xylB</i> the activity of <i>xylB</i> could been proven</p>
 +
<p align="justify">Our cells were again inoculated and induced at an OD of 0.6. This time we added <small>D</small>-xylose at an concentration of 4g/l. After induction for 12 hours cells were harvested and lysated. The cell lysate was chemically extracted with dichlormethan and analysed with HPLC-MS. Unfortunately in our measurement no ethylene glycol could be verified. It is possible that overexpression of the other enzymes of the pathway is necessary for significant production in <i>E. coli</i>.</p>
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<br>
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    <table>
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        <colgroup width=50%>
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        <tr>
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            <td align= "center" valign="middle">
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                <img class="transparent" alt="https://static.igem.org/mediawiki/parts/8/8e/TU_Darmstadt_EG_xylBC_PAGE.png" src="https://static.igem.org/mediawiki/parts/8/8e/TU_Darmstadt_EG_xylBC_PAGE.png">
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<tr>
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<td align="left">
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                <div><b>Figure 2</b> <p align="justify">Scan of the PAGE containing four different samples: marker (M; Protein Marker III AppliChem); samples 1-4 reference and induced.</p></div>
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            </td>
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</tr>
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<td align= "center" valign="middle">
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                <img class="shrinkToFit" alt="https://static.igem.org/mediawiki/parts/9/9a/T7-xylB-xylC_Assay.png" src="https://static.igem.org/mediawiki/parts/9/9a/T7-xylB-xylC_Assay.png" height="432" width="740">
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</tr>
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            </td>
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<td align= "center" valign="middle">
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                <img class="shrinkToFit transparent" alt="https://static.igem.org/mediawiki/parts/4/4f/Microplate_assay_XylB.png" src="https://static.igem.org/mediawiki/parts/4/4f/Microplate_assay_XylB.png" height="407" width="684"> </td>
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</tr>
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            <td align="left">
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                <div><b>Figure 3</b> Plot of the NAD<sup>+</sup> assay. <i>xylB</i> shows activity</div>
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            </td>
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<td align= "bottom" valign="left">
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                <div><img class="transparent" alt="<img class="shrinkToFit transparent" alt="https://static.igem.org/mediawiki/parts/4/44/TU_Darmstadt_MS_mit_EG.png" src="https://static.igem.org/mediawiki/parts/4/44/TU_Darmstadt_MS_mit_EG.png" height="407" width="651">"></div>
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</td>
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</tr>
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<tr>
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<td> <b>Figure 4:</b> HPLC-MS spectra from cell lysate with artificialy added ethylene glycol
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</td>
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</tr>
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<br>
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            <td align= "bottom" valign="left">
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                <div><img class="shrinkToFit transparent" alt="https://static.igem.org/mediawiki/parts/4/40/TU_Darmstadt_MS_ohne_EG.png" src="https://static.igem.org/mediawiki/parts/4/40/TU_Darmstadt_MS_ohne_EG.png" height="407" width="651"></div>
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            </td>
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<tr>
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<td>
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<br>
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<b>Figure 5:</b> Our culture which was induced with xylose showed no ethyleneglycol after extraction in mass spectrometrie</td>
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</tr>
 +
        </tr>
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 +
        </colgroup>
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    </table>
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    <div>
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<br>
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</html>
  
 
===<h2>Sequence and Features</h2>===
 
===<h2>Sequence and Features</h2>===
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<partinfo>BBa_K1602005 SequenceAndFeatures</partinfo>
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<partinfo>BBA_K1602017 SequenceAndFeatures</partinfo>
 +
 
 +
<br>
 +
===References===
 +
 
 +
1. Liu H, Ramos KR, Valdehuesa KN, Nisola GM, Lee WK, Chung WJ. Biosynthesis of ethylene glycol in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(8):3409-17.
 +
 
 +
2. Toivari MH, Nygard Y, Penttila M, Ruohonen L, Wiebe MG. Microbial D-xylonate production. Appl Microbiol Biotechnol. 2012;96(1):1-8.

Latest revision as of 03:38, 19 September 2015

D-xylonic acid producing operon

D-Xylose is a monosaccharide belonging to the aldopentose family. It was recently shown that the D-xylose dehydrogenase xylB from Caulobacter crescentus can convert D-xylose to D-xylonolactone. This can react spontaneously or through the catalysation of xylC to D-xylonic acid. (2)

In E. coli D-xylonic acid can further be metabolized to ethyleneglycol by the enzymes yjhG (BBa_K1602012), yagE (BBa_K1602011) and yqhD (BBa_K1602013) which are already present in this host. (1)


https://static.igem.org/mediawiki/parts/f/f1/TU_Darmstadt_EG_XylB-xylC.png

  • Figure 1 Sheme of the reactions catalyzed by xylB and xylC. The xylC reaction can also happen spontaneously but in a much lower speed. (2)



    • Usage

    This part is a composite of two coding genes with strong RBS (BBa_B0034). The transcription is controlled by a T7 promotor (BBa_I719005).

    We used this operon to investigate possible production of ethylene glycol in E. coli.


    https://static.igem.org/mediawiki/parts/1/12/TU_Darmstadt_Plasmidkarte_T7-B0034-xylB-B0034-xylC.png


    Results

    E. coli BL21 were transformed with the operon and grown to a OD of 0.6. A negative sample was taken before IPTG was added to a concentration of 1mM for induction. Cells stayed at 28°C for 12 hours and later were harvested and resuspended in buffer. A small amount of both induced samples and negative samples was loaded on a SDS-PAGE while proteins were extracted from the rest. The SDS-PAGE showed overexpression of proteins of the expected mass.

    In a NAD+ assay activity of xylB the activity of xylB could been proven

    Our cells were again inoculated and induced at an OD of 0.6. This time we added D-xylose at an concentration of 4g/l. After induction for 12 hours cells were harvested and lysated. The cell lysate was chemically extracted with dichlormethan and analysed with HPLC-MS. Unfortunately in our measurement no ethylene glycol could be verified. It is possible that overexpression of the other enzymes of the pathway is necessary for significant production in E. coli.









    https://static.igem.org/mediawiki/parts/8/8e/TU_Darmstadt_EG_xylBC_PAGE.png
    Figure 2

    Scan of the PAGE containing four different samples: marker (M; Protein Marker III AppliChem); samples 1-4 reference and induced.
    https://static.igem.org/mediawiki/parts/9/9a/T7-xylB-xylC_Assay.png
    https://static.igem.org/mediawiki/parts/4/4f/Microplate_assay_XylB.png
    Figure 3 Plot of the NAD+ assay. xylB shows activity
    <img class=">
    Figure 4: HPLC-MS spectra from cell lysate with artificialy added ethylene glycol

    https://static.igem.org/mediawiki/parts/4/40/TU_Darmstadt_MS_ohne_EG.png

    Figure 5: Our culture which was induced with xylose showed no ethyleneglycol after extraction in mass spectrometrie

    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
      COMPATIBLE WITH RFC[25]
    • 1000
      COMPATIBLE WITH RFC[1000]


    References

    1. Liu H, Ramos KR, Valdehuesa KN, Nisola GM, Lee WK, Chung WJ. Biosynthesis of ethylene glycol in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(8):3409-17.

    2. Toivari MH, Nygard Y, Penttila M, Ruohonen L, Wiebe MG. Microbial D-xylonate production. Appl Microbiol Biotechnol. 2012;96(1):1-8.