Difference between revisions of "Part:BBa K1602017"
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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 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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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>.
 
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Revision as of 02:42, 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.
    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 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.
    Figure 3 Plot of the gel lanes based on contrast analyses - created with ImageJ
    <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.