Difference between revisions of "Part:BBa K1602017"
 
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===<h2>Results</h2>===
 
===<h2>Results</h2>===
 
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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.
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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>
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<p align="justify">In a NAD<sup>+</sup> assay activity of <i>xylB</i> the activity of <i>xylB</i> could been proven</p>
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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 production in <i>E. coli</i>.</p>
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<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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                 <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">
 
                 <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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                <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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                <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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                <div><img style="width: 300px; height: 456;" src="https://static.igem.org/mediawiki/2015/a/a7/Da15_st.png" href="http://2015._sds_ cadA_plot.png"</div>
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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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                 <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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                 <div><b>Figure 3</b> Plot of the NAD<sup>+</sup> assay. <i>xylB</i> shows activity</div>
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                <div><b>Figure 3</b> Plot of the gel lanes based on contrast analyses - created with ImageJ</div>
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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.